Titration of low K(d) binding sites: binding of arginine analogs to nitric oxide synthases.

Spectrophotometrically monitored ligand titration is an important method for the determination of equilibrium dissociation constants (K(d)) from nitric oxide synthases (NOS). Low K(d) sites such as the tetrahydrobiopterin and arginine binding sites present difficulties in that experiments often require enzyme concentrations of the same magnitude as the K(d). An analytical method based on computer simulation is described that allows the estimation of K(d) values without an independent means of monitoring free ligand or without an accurate prior determination of the number of binding sites. The K(d) for arginine is approximately 0.5 microM for the tetrahydrobiopterin replete neuronal and inducible isoforms (nNOS and iNOS), while the endothelial isoform has a slightly higher K(d) (1.5 microM). N-OH-arginine (an intermediate) binds to nNOS with a K(d) of around 0.2 microM, while the inhibitors N-methyl-arginine and N-nitro-arginine bind more tightly; our best K(d) estimates are 100 nM or lower.     

A simple method for calculating the dissociation constant of a receptor (or enzyme).unlabeled ligand complex from radioligand displacement measurements

A general procedure is described for determining the dissociation constant of a receptor (or enzyme).unlabeled ligand complex (EI) by analyzing the I-dependent displacement of bound radioligand (A*) from EA*. The procedure (which involves measuring free A* in the presence of I) requires a knowledge of the total concentrations of receptor ([E]t), unlabeled ligand ([I]t) and radioligand ([A*]t), and the dissociation constant of the EA* complex. The unknown Kd is obtained from five simple, sequential calculations which are valid for either high or low affinity competitive unlabeled ligands and are independent of total receptor concentration or initial degree of saturation with A*. The procedure also provides the information needed to construct a distribution curve of all enzyme and ligand species (E, EA*, EI, A*, I) as [I]t is varied.     

The N-terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two-step binding by rapid-kinetic studies of cystatin A labeled at the N-terminus with a fluorescent reporter group

The three-dimensional structures of cystatins, and other evidence, suggest that the flexible N-terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two-step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N-terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS- and AEDANS-labeled cystatin A to papain resulted in approximately 4-fold and 1.2-fold increases of probe fluorescence, respectively, reflecting the interaction of the N-terminal region with the enzyme. Observed pseudo-first-order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS-cystatin A increased linearly with the concentration of the latter. In contrast, pseudo-first-order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N-terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two-step manner. Such independent binding of the N-terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin-proteinase reactions.     

A fluorescence resonance energy transfer method for measuring the binding of inhibitors to stromelysin.

A sensitive fluorescence resonance energy transfer method was developed for the direct measurement of the dissociation constants of stromelysin inhibitors. The method is applied to the thiadiazole class of stromelysin inhibitors and it takes advantage of the fact that, upon binding to the active site of enzyme, the thiadiazole ring, with its absorbance centered at 320 nm, is able to quench the fluorescence of the tryptophan residues surrounding the catalytic site. The changes in fluorescence are proportional to the occupancy of the active site: Analysis of the fluorescence versus inhibitor concentration data yields dissociation constants that are in agreement with the corresponding competitive inhibitory constants measured by a catalytic rate assay. The affinity of nonthiadiazole inhibitors of stromelysin-such as hydroxamic acids and others-can be determined from the concentration-dependent displacement of a thiadiazole of known affinity. Using this displacement method, we determined the affinities of a number of structurally diverse inhibitors toward stromelysin. Since the three tryptophan residues located in the vicinity of the active site of stromelysin are conserved in gelatinase and collagenase, the method should also be applicable to inhibitors of other matrix metalloproteinases.     

Trypsin inhibitor in the hemolymph of a solitary ascidian, Halocynthia roretzi. Purification and characterization

A purified preparation of trypsin inhibitor was obtained from the hemolymph of a solitary ascidian, Halocynthia roretzi, by a procedure including trypsin-Sepharose chromatography, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The product was a mixture of two isoinhibitors, inhibitors I and II. They were separated from each other by high-performance liquid chromatography on an anion exchanger column, and showed almost identical amino acid compositions. They were also indistinguishable in terms of apparent specific inhibitory activity against bovine trypsin when the activity was assayed with the inhibitors at rather high concentrations (greater than 50 nM). A large difference was observed between them, however, in the inhibition constants, which correspond to the dissociation constants of the inhibitor-trypsin complexes; the inhibition constant of inhibitor I was 90 pM, whereas that of inhibitor II was 4.7 nM. The molecular weights of inhibitors I and II were estimated to be 6,000 and 4,500, respectively, by SDS-polyacrylamide gel electrophoresis, while an almost identical value, 9,000, was obtained for both of them by gel filtration. The molecular weight calculated from the amino acid compositions was 5,929 for both. The isoelectric points were also identical, that is about 5.0. Both of the inhibitors were heat-stable. Ascidian inhibitor I also inhibited other trypsin-like enzymes of mammalian origin, as well as those of ascidian origin.     

Interaction of mouse submaxillary gland renin with a statine-containing, subnanomolar, competitive inhibitor

The interaction between mouse submaxillary gland renin and a statine-containing, iodinated substrate analog inhibitor was studied. The compound, 1 (Boc-His-Pro-Phe-(4-iodo)-Phe-Sta-Leu-Phe-NH2, Sta = (3S,4S)-4-amino-3-hydroxy-6-methyl-heptanoic acid), a statine-containing analog of the renin substrate octapeptide, was a competitive inhibitor of cleavage of synthetic tetradecapeptide renin substrate by mouse submaxillary gland renin, with a Ki of 6.2 x 10(-10) M (pH 7.2, 37 degrees C). Titration of the partial quenching of the tryptophan fluorescence of the enzyme by 1 revealed tight binding with a dissociation constant less than 3 nM and a binding stoichiometry of one mole 1 per mole enzyme. The time course of tight binding of 1 to mouse renin appeared to be fast, with kON greater than or equal to 1.3 x 10(6) s-1 M-1. The UV difference spectrum generated upon binding of 1 to mouse renin had two prominent features: a strong, broad band that had a minimum at 242 nm with delta epsilon (242) = -19,500 cm-1 M-1, and a triplet of enhanced bands centered at 286 nm with delta epsilon (286) about +1100 cm-1 M-1. The strong, broad, negative band was similar to the difference between the UV absorbance of 1 in methanol and in 0.1 M citrate phosphate pH 7.2. A structure-activity correlation for analogs of 1 showed some moieties of 1 that are important for potent inhibition of mouse renin. The inhibition data for these compounds versus human kidney renin suggested that the solution of the crystal structure of 1 bound to mouse renin will provide useful information for the design of inhibitors of human kidney renin.     

Determination of interaction kinetic constants for HIV-1 protease inhibitors using optical biosensor technology

The interaction between HIV-1 protease and inhibitors has been studied with optical biosensor technology. Optimized experimental procedures and mathematical analysis permitted determination of association and dissociation rate constants. A sensor surface with native enzyme was unstable and exhibited a drift that was influenced by the binding of inhibitor. This was hypothesized to be due to a specific mechanism involving autoproteolysis and/or dimer dissociation. The use of a mutant predicted to be less susceptible to autoproteolysis (Q7K) than wild-type enzyme resulted in a minor effect on surface stability, while a completely stable surface was obtained by treatment of the immobilized enzyme with N-ethyl-N'-(dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide; the most stable surface was achieved by chemically modifying the Q7K enzyme. The stabilized surface was enzymatically active and the interaction with inhibitors was similar to that for native enzyme. Several of the inhibitors had very high association rates, and estimation of kinetic constants was therefore performed with a binding equation accounting for limited mass transport. Of the clinical inhibitors studied, saquinavir had the highest affinity for the enzyme, a result of the lowest dissociation rate. Although the dissociation rate for ritonavir was sixfold faster, the affinity was only twofold lower than that for saquinavir since the association rate was threefold faster. Nelfinavir and indinavir exhibited lower affinities relative to the other inhibitors, a consequence of a slower association for nelfinavir and a relatively fast dissociation for indinavir. These results show that biosensor-based interaction studies can resolve affinity into association and dissociation rates, and that these are characteristic parameters for the interaction between enzymes and inhibitors.     

Biochemical characterization of TAK-593, a novel VEGFR/PDGFR inhibitor with a two-step slow binding mechanism

Inhibition of tumor angiogenesis leads to a lack of oxygen and nutrients in the tumor and therefore has become a standards of care for many solid tumor therapies. Dual inhibition of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) protein kinase activities is a popular strategy for targeting tumor angiogenesis. We discovered that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, potently inhibits tyrosine kinases from the VEGFR and PDGFR families. TAK-593 was highly selective for these families, with an IC(50) >1 μM when tested against more than 200 protein and lipid kinases. TAK-593 displayed competitive inhibition versus ATP. In addition, TAK-593 inhibited VEGFR2 and PDGFRβ in a time-dependent manner, classifying it as a type II kinase inhibitor. Analysis of enzyme-inhibitor preincubation experiments revealed that the binding of TAK-593 to VEGFR2 and PDGFRβ occurs via a two-step slow binding mechanism. Dissociation of TAK-593 from VEGFR2 was extremely slow (t(1/2) >17 h), and the affinity of TAK-593 at equilibrium (K(i)*) was less than 25 pM. Ligand displacement analysis with a fluorescent tracer confirmed the slow dissociation of TAK-593. The dissociation rate constants were in good agreement between the activity and ligand displacement data, and both analyses supported slow dissociation of TAK-593. The long residence time of TAK-593 may achieve an extended pharmacodynamic effect on VEGFR2 and PDGFRβ kinases in vivo that differs substantially from its observed pharmacokinetic profile.     

Exploring the substructural space of indole-3-carboxamide derivatives binding to renin: a novel active-site spatial partitioning approach

Renin has recently attracted much attention in the antihypertensive community, since this enzyme starts the angiotensin-converting cascade and forms the rate-limiting step in this cascade. In the present study, we describe a new method called active-site spatial partitioning (ASSP) for quantitatively characterizing the nonbonding interaction profile between renin and the substructures of indole-3-carboxamide derivatives-a novel class of achiral renin inhibitors that exhibit both high affinity and strong specificity for renin, thus blocking its active state-on the basis of structural models of protein-ligand complexes. It is shown that the ASSP-derived potential parameters are highly correlated with the experimentally measured activities of indole-3-carboxamides; the statistical models linking the parameters and activities using a sophisticated partial least squares regression technique show much promise as an effective and powerful tool for generalizing and predicting the pharmaceutical potencies and the physicochemical properties of other modified derivatives. Furthermore, by visually examining substructure-color plots generated by the ASSP procedure, it is found that the relative importance of nonbonding contributions to the recognition and binding of a ligand by renin is as follows: steric < hydrophobic < electrostatic. The polar and charged moieties that float on the surface of the ligand molecule play a critical role in conferring electrostatic stability and specificity to renin-ligand complexes, whereas the aromatic rings embedded in the core region of the ligand are the main source of hydrophobic and steric potentials that lead to substantial stabilization of the complex architecture.     

Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.     

Characterization of inhibitor binding sites of mitochondrial complex I using fluorescent inhibitor

Recent progress in complex I research suggests that a wide variety of complex I inhibitors share a common large binding domain with partially overlapping sites. To verify this concept, we carried out real-time displacement tests of a fluorescent ligand with various competitors using a novel quinazoline-type inhibitor (aminoquinazoline, AQ). In the presence of an excess amount of the competitors, the binding of AQ to the enzyme was completely suppressed, being in line with the concept mentioned above. However, AQ bound to the enzyme was not displaced by subsequent addition of an increasing amount of competitors in the concentration range expected from the relative magnitude of the K(d) values of AQ and competitors, rather, much higher concentrations of the competitors were needed to displace bound AQ. These results cannot be explained merely by the premise of a common or partially overlapping binding site(s) between AQ and competitors. On the other hand, double-inhibitor titration of steady state complex I activity suggested that additivity of inhibition is not necessarily observed for all pairs of complex I inhibitors. Our results are discussed in light of the cooperativity of the inhibitor binding sites.     

Inhibition of wild-type and mutant human immunodeficiency virus type 1 proteases by GW0385 and other arylsulfonamides

The arylsulfonamide derivatives described herein were such potent inhibitors of human immunodeficiency virus type 1 (HIV-1) protease (enzyme, E) that values for the inhibition constants (K(i)) could not be determined by conventional steady-state kinetic techniques (i.e., the minimal enzyme concentration usable for the activity assay was much greater than the value of the dissociation constant). Consequently, two alternative methods were developed for estimation of K(i) values. The first method employed kinetic determinations of values for k(1) and k(-1), from which K(i) was determined (k(-1)/k(1)). The second method was a competitive displacement assay used to determine binding affinities of other inhibitors relative to that of GW0385. In these assays, the inhibitor of unknown affinity was used to displace [(3)H]GW0385 from E.[(3)H]GW0385. From the concentration of E.[(3)H]GW0385 at equilibrium, the concentrations of enzyme-bound and free inhibitors were calculated, and the ratio of the K(i) value of the unknown to that of GW0385 was determined (K(i,unknown)/K(i,GW0385)). The values of k(1) were calculated from data in which changes in the intrinsic protein fluorescence of the enzyme associated with inhibitor binding were directly or indirectly monitored. In the case of saquinavir, the fluorescence changes associated with complex formation were large enough to monitor directly. The value of k(1) for saquinavir was 62 +/- 2 microM(-1) s(-1). In the case of GW0385, the fluorescence changes associated with complex formation were too small to monitor directly. Consequently, the value of k(1) was estimated from a competition experiment in which the effect of GW0385 on the binding of E to saquinavir was determined. The value of k(1) for GW0385 was estimated from these experiments to be 137 +/- 4 microM(-1) s(-1). Because E.[(3)H]GW0385 was stable in the standard buffer at room temperature for greater than 33 days, the value of the first-order rate constant for dissociation of E.[(3)H]GW0385 (k(-1)) could be estimated from the time-course for exchange of E.[(3)H]GW0385 with excess unlabeled GW0385. The value of k(-1) calculated from these data was (2.1 +/- 0.1) x10(-6) s(-1) (t(1/2) = 91 h). The K(i) value of wild-type HIV-1 protease for GW0385, calculated from these values for k(1) and k(-1), was 15 +/- 1 fM. Three multidrug resistant enzymes had K(i) values for GW0385 that were less than 5 pM.     

Characterization of the receptor to vasculotropin on bovine adrenal cortex-derived capillary endothelial cells

Recently a new growth factor was purified to homogeneity, and its bioactivity seemed to be restricted to vascular endothelial derived cells. As it was also angiogenic in vivo, it was provisionally named vasculotropin (VAS). As an iodination procedure used to label VAS did not damage the molecule, it was possible to undertake binding studies. The binding of iodinated vasculotropin to bovine adrenal cortex-derived capillary endothelial cells was saturable at 250 pM, and half-maximal binding occurred at 47 pM. Scatchard's analysis of the data demonstrated two apparent classes of binding sites with apparent dissociation constants of 2 and 82 pM displaying 280 and 3400 binding sites, respectively. The binding was specific; half-displacement was observed with a 2-fold excess of unlabeled VAS. The structurally related platelet-derived growth factor did not compete in a radioreceptor assay. 125I-VAS was displaced by suramin and not by heparin. 125I-VAS was covalently cross-linked to its cell surface receptor on intact bovine adrenal cortex-derived capillary endothelial cells using the homobifunctional agents ethylene glycol bis(succinimidyl succinate) or disuccinimidyl tartarate. A major macromolecular species with an apparent molecular mass of 230,000 Da was labeled under reducing and nonreducing conditions. These data demonstrate the existence of a specific binding protein for VAS and an estimation of the size at 185,000 Da.     

Inhibition of fructose-1,6-bisphosphate aldolase from rabbit muscle and Bacillus stearothermophilus

Phosphoglycollohydroxamic acid and phosphoglycollamide are inhibitors of rabbit muscle fructose-1,6-bisphosphate aldolase. The binding dissociation constants determined by enzyme inhibition and protein fluorescence quenching suggest that two distinct enzyme inhibitor complexes may be formed. The binding dissociation constants of the two inhibitors to Bacillus stearothermophilus cobalt (II) fructose-1,6-bisphosphate aldolase have also been determined. The hydroxamic acid is an exceptionally potent inhibitor (Ki = 1.2 nM) probably due to direct chelation with Co(II) at the active site. The inhibition, however, is time-dependant and the association and dissociation constants have been estimated. Ethyl phosphoglycollate irreversibly inhibits rabbit muscle fructose-1,6-bisphosphate aldolase in the presence of sodium borohydride, presumably by forming a stable secondary amine through the active-site lysine reside. A new condensation assay for fructose-1,6-bisphosphate aldolases has been developed which is more sensitive than currently used assay procedures.     

Kinetic evidence for an activation step following binding of human interferon alpha 2 to the membrane receptors of Daudi cells

A single species of human interferon alpha (IFN alpha) was labelled with 125I to high incorporation for binding studies on the B-lymphoblastoid cell line, Daudi, whose growth is inhibited by low doses of IFN, the effect being saturated at about 100 U/ml (25 pM). The radiolabelled IFN was shown to be fully active and the binding affinity to cellular sites was shown to be unchanged by iodination. Experimental conditions were standardized such that binding and cell growth experiments could be performed on the same initial culture of cells. 125I-labelled IFN alpha 2 (IFN alpha prepared from Escherichia coli carrying human alpha 2 gene) was added to exponentially growing cultures (mean specific growth rate 0.77 +/- 0.07 days-1) at a mean concentration of 235000 +/- 20000 cells ml-1. Two types of binding could be discerned on growing cultures: the first with a transient peak followed by a loss or discharge of available sites, the second reaching equilibrium some 3 h after the addition of IFN. Large differences in the apparent dissociation constants were evident. The affinity of binding at the 'steady-state', appeared to be much higher. An analysis of the displacement rates for bound IFN suggested that the two reactions were occurring consecutively over the whole of the dose range studied (1-100 U/ml; 0.25-25 pM IFN). In this dose range we found that Daudi cells would eventually stop growing at all doses and that the rates of deceleration of cellular growth were linearly proportional to the dose of IFN in a double-reciprocal plot (i.e. in analogy to Michaelis-Menten kinetics). A good congruence was found between the equilibrium constants for binding and for growth inhibition (2.65 pM and 2.39 pM, respectively). The amount of IFN bound at steady state thus determines the rate at which growth is inhibited. We propose that the first reaction represents binding of IFN to surface receptors, and the second transfer of IFN to an activation complex on the cell membrane. Appropriate models and their general applicability to IFN action are discussed.     

Hydroxyurea is a carbonic anhydrase inhibitor

The interaction of hydroxyurea with the cytosolic isozymes of carbonic anhydrase (CA), hCA I and hCA II has been investigated by means of kinetic and spectroscopic techniques. Hydroxyurea acts as a weak, non-competitive inhibitor of both isozymes, for the 4-nitrophenyl acetate esterase activity, with inhibition constants around 0.1 mM for both isozymes. The spectrum of the adduct of hydroxyurea with Co(II)-hCA II is similar to the spectra of tetrahedral adducts (such as those with sulfamide, acetazolamide or cyanamide), proving a direct interaction of the inhibitor molecule with the metal center of the enzyme, whose geometry remains tetrahedral. Based on the X-ray crystal structure of the adducts of hCA II with ureate and hydroxamate inhibitors, the hypothetical binding of hydroxyurea is proposed to be achieved in deprotonated state, with the nitrogen atom coordinated to Zn(II), and the OH group of the inhibitor making a hydrogen bond with Thr 199. This binding may be exploited for the design of both CA as well as matrix metalloproteinase (MMP) inhibitors, since hydroxyurea is the simplest compound incorporating a hydroxamate functionality in its molecule. Indeed, such inhibitors of the sulfonylated amino acid hydroxamate type have been generated, with potencies in the low nanomolar range for both type of enzymes, CAs and MMPs.     

Analysis of the interaction between the aspartic peptidase inhibitor SQAPI and aspartic peptidases using surface plasmon resonance

Aspartic peptidase inhibitors, which are themselves proteins, are strong inhibitors (small inhibition constants) of some aspartic peptidases but not others. However, there have been no studies of the kinetics of the interaction between a proteinaceous aspartic peptidase inhibitor and aspartic peptidases. This paper describes an analysis of rate constants for the interaction between recombinant squash aspartic peptidase inhibitor (rSQAPI) and a panel of aspartic peptidases that have a range of inhibition constants for SQAPI. Purified rSQAPI completely inhibits pepsin at a 1:1 molar ratio of pepsin to rSQAPI monomer (inhibition constant 1 nM). The interaction of pepsin with immobilized rSQAPI, at pH values between 3.0 and 6.0, was monitored using surface plasmon resonance. Binding of pepsin to rSQAPI was slow (association rate constants ca 10(4)M (-1)s(-1)), but rSQAPI was an effective pepsin inhibitor because dissociation of the rSQAPI-pepsin complex was much slower (dissociation rate constants ca 10(-4)s(-1)), especially at low pH values. Similar results were obtained with a His-tagged rSQAPI. Strong inhibition (inhibition constant 3 nM) of one isoform (rSap4) of the family of Candida albicans-secreted aspartic peptidases was, as with pepsin, characterized by slow binding of rSap4 and slower dissociation of the rSap4-inhibitor complex. In contrast, weaker inhibition of the Glomerella cingulata-secreted aspartic peptidase (inhibition constant 7 nM) and the C. albicans rSap1 and Sap2 isoenzymes (inhibition constants 25 and 400 nM, respectively) was, in each case, characterized by a larger dissociation rate constant.     

Towards the mechanism of trimeric purine nucleoside phosphorylases: stopped-flow studies of binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine

The binding of multisubstrate analogue inhibitor - 2-amino-9-[2-(phosphonomethoxy)ethyl]-6-sulfanylpurine (PME-6-thio-Gua) to purine nucleoside phosphorylase from Cellulomonas sp. at 20 degrees C, in 20 mM Hepes buffer with ionic strength adjusted to 50 mM using KCl, at several pH values between 6.5 and 8.2, was investigated using a stopped-flow spectrofluorimeter. The kinetic transients registered after mixing a protein solution with ligand solutions of different concentrations were simultaneously fitted by several association reaction models using nonlinear least-squares procedure based on numerical integration of the chemical kinetic equations appropriate for given model. It is concluded that binding of a PME-6-thio-Gua molecule by each of the binding sites is sufficiently well described by one-step process, with a model assuming interacting binding sites being more probable than a model assuming independent sites. The association rate constants derived from experimental data, assuming one step binding and independent sites, are decreasing with an increase in pH, changing from 30 to 6 microM(-1)s(-1) per binding site. The dissociation rate constants are in the range of 1-3 s(-1), and they are rather insensitive of changes in pH. Interestingly, for each pH value, the one-step binding model with interacting sites results in the association rate constant per site 1.5-4 times smaller for the binding of the first ligand molecule than that for the binding of the second one. Decrease of association constants with pH indicate that the enzyme does not prefer binding of the naturally occurring anionic form of the 6-thioguanine ring (pK(a) 8.7) resulting from a dissociation of N(1)-H. This finding supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 (Glu 201 in mammalian PNPs) is crucial in the catalytic process. Results obtained also indicate that, in contrast to transition-state analogues, for which binding is followed by a conformational change, binding of multisubstrate analogue inhibitors to trimeric PNPs is a one-step process.     

IL-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts.

IL-12 is a 75-kDa heterodimeric cytokine composed of disulfide-bonded 35-kDa and 40-kDa subunits. Included among the biologic activities mediated by IL-12 is induction of proliferation of PHA-activated human PBL. The concentration of IL-12 required to stimulate maximum proliferation of PHA-activated lymphoblasts is 50 to 100 pM. In this study, highly purified 125I-labeled IL-12 (7 to 15 microCi/microgram; 50 to 100% bioactive) was used to characterize the receptor for IL-12 on 4-day PHA-activated lymphoblasts. The binding of 125I-labeled IL-12 to PHA-activated lymphoblasts was saturable and specific because the binding of radiolabeled ligand was only inhibited by IL-12 and not by other cytokines. The kinetics of [125I]IL-12 binding to PHA-activated lymphoblasts was rapid at both 4 degrees C and 22 degrees C; reaching equilibrium within 60 min. At 22 degrees C, the rate of dissociation of [125I]IL-12 was slow in the absence of competing IL-12 (t1/2 = 5.9 h) and more rapid in the presence of 25 nM competing IL-12 (t1/2 = 2.5 h). The kinetically derived equilibrium dissociation constant ranged from 10 to 83 pM. Analysis of steady state binding data by the method of Scatchard identified a single binding site with an apparent equilibrium dissociation constant of 100 to 600 pM and 1000 to 9000 sites/lymphoblast. The equilibrium dissociation constant for competing ligands and sites per cell calculated from unlabeled IL-12 competition experiments ranged from 164 to 315 pM and 1067 to 3336, respectively, which is in good agreement with the values determined from steady state binding. The variations in KD and sites per cell were dependent on the individual preparations of lymphoblasts. Although the steady state binding data were consistent with a single class of high affinity binding sites, the kinetic dissociation data indicates a cooperative interaction between receptors on PHA-activated lymphoblasts. Affinity cross-linking of surface bound [125I]IL-12 to PHA-activated lymphoblasts at 4 degrees C identified a major complex of approximately 210 to 280 kDa. Anti-IL-12 antibodies also immunoprecipitated a complex of approximately 210 to 280 kDa that was produced by cross-linking unlabeled IL-12 to 125I-labeled lymphoblast cell-surface proteins. Cleavage of this complex with reducing agent identified one radiolabeled protein of approximately 110 kDa. These data suggest that the IL-12 binding site on PHA-activated lymphoblasts may be composed of a single protein of approximately 110 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)     

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.