Determination Of Inhibition Constants Research Articles

En Route to New Therapeutic Options for Iron Overload Diseases: Matriptase-2 as a Target for Kunitz-Type Inhibitors.

The cell-surface serine protease matriptase-2 is a critical stimulator of iron absorption by negatively regulating hepcidin, the key hormone of iron homeostasis. Thus, it has attracted much attention as a target in primary and secondary iron overload diseases. Here, we have characterised Kunitz-type inhibitors hepatocyte growth factor activator inhibitor 1 (HAI-1) and HAI-2 as powerful, slow-binding matriptase-2 inhibitors. The binding modes of the matriptase-2-HAI complexes were suggested by molecular modelling. Different assays, including cell-free and cell-based measurements of matriptase-2 activity, determination of inhibition constants and evaluation of matriptase-2 inhibition by analysis of downstream effects in human liver cells, demonstrated that matriptase-2 is an excellent target for Kunitz inhibitors. In particular, HAI-2 is considered a promising scaffold for the design of potent and selective matriptase-2 inhibitors.

Pim Kinase Inhibitors Evaluated with a Single‐Molecule Engineered Nanopore Sensor

AbstractProtein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label‐free determination of inhibition constants. The procedure circumvents many sources of bias or false‐positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips.

Identification of VKORC1 genotype leading to resistance to tecarfarin.

The VKORC1 gene codes for the VKORC1 enzyme that is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). The use of VKA in human medicine is difficult because of inter-individual variability in VKA requirements. This variability is partly due to basic physiological parameters but also to genetic polymorphisms in CYP2C9 involved in VKA metabolism. Tecarfarin, a new VKA, was developed to avoid the CYP2C9-dependent metabolism. This molecule is metabolized by carboxylesterases. Nevertheless, genetic variations in the VKORC1 gene are also associated with inter-individual variability in VKA requirements. This study was performed to estimate the benefit of using tecarfarin versus traditional VKA in human patient carrier for hVKORC1 mutation. Wild type hVKORC1 and its spontaneous mutants were expressed in Pichia pastoris and susceptibility to tecarfarin was assessed by the in vitro determination of inhibition constants. Among the 14 mutated VKORC1 proteins analyzed in this study, 6 mutations (i.e., A26P, A41S, V54L, H68Y, I123N, and Y139H) led to resistant to traditional VKAs, while only 4 mutations (i.e., A26P, V54L, I123N, and Y139H) led to resistant to tecarfarin. Compared to other VKAs, a clear benefit of tecarfarin is observed for the mutants A26P, A41S, H68Y, and I123N and an absence of benefit for the mutant Y139H. Finally, tecarfarin is particularly inefficient on V54L-VKORC1.

A Modular Strategy to Prepare Multivalent Inhibitors of Prostate-Specific Membrane Antigen (PSMA)

We have developed a modular scaffold for preparing high-affinity, homo-multivalent inhibitors of the prostate-specific membrane antigen (PSMA) for imaging and therapy of prostate cancer (PCa). Our system contains a lysine-based (µ-, e-) dialkyne residue for incorporating a PSMA binding Lys-Glu urea motif exploiting click chemistry and a second lysine residue for subsequent modification with an imaging or therapeutic moiety. The utility of the multivalent scaffold was examined by synthesizing bivalent compounds 2 and 3 and comparing them with the monovalent analog 1. Determination of inhibition constants (Ki) revealed that bivalent 2 (0.2 nM) and 3 (0.08 nM) are significantly more potent (~ 5 fold and ~ 11 fold, respectively) inhibitors of PSMA than monovalent 1 (0.9 nM). A single photon emission computed tomography (SPECT)-CT imaging study of [111In]3 demonstrated high and specific uptake in PSMA+ PC-3 PIP tumor until at least 48 h post-injection, with rapid clearance from non-target tissues, including kidney. A biodistribution study revealed that [111In]3 demonstrated 34.0 ± 7.5 percent injected dose per gram of tissue in PSMA+ tumor at 24 h post-injection and was capable of generating target-to-non-target ratios of ~ 379 in PSMA+ PC-3 PIP tumors vs. isogenic PSMA-negative PC3-flu tumors in vivo. The click chemistry approach affords a convenient strategy toward multivalent PSMA inhibitors of enhanced affinity and superior pharmacokinetics for imaging.

Magnetic “Fishing” Assay To Screen Small-Molecule Mixtures for Modulators of Protein−Protein Interactions

Protein-protein interactions are an intricate part of biological pathways and have become important targets for drug discovery. Here we present a two-stage magnetic bead assay to functionally screen small-molecule mixtures for modulators of protein-based interactions, with simultaneous affinity-based isolation of active compounds and identification by mass spectrometry. Proteins of interest interact in solution prior to the addition of Ni(II)-functionalized magnetic beads to recover an intact protein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing"). Protein-complex fishing, utilizing His(6)-tagged calmodulin (CaM) as the primary (bait) protein and melittin (Mel) as the target, was used to screen a mass-encoded library of 1000 bioactive compounds (50 mixtures, 20 compounds each) and successfully identified three known antagonists, three naturally occurring phenolic compounds previously reported to disrupt CaM-activated phosphodiesterase activity, and two newly identified modulators of the CaM-Mel interaction, methylbenzethonium and pempidine tartrate. The ability to produce quantitative inhibition data is also shown through the development of dose-dependent response curves and the determination of inhibition constants (K(I)) for the novel compound methylbenzethonium (K(I) = 14-49 nM) and two known antagonists, calmidazolium (K(I) = 1.7-7.5 nM) and trifluoperazine (K(I) = 1.2-3.0 μM), with the latter two values being in close agreement with literature values.

Selective inhibitors of plasmepsin II of Plasmodium falciparum on the basis of pepstatin

A number of new inhibitors of plasmepsin II (PlmII) from Plasmodium falciparum, one of the key factors of malarial parasite survival, were synthesized. The inhibitors are analogues of pepstatin with various variants of Ala residue substitutions. Effects of the inhibitors on human PlmII and cathepsin D were studied. Inhibition of PlmII by the substrate was found, which required the use of the modified Henderson method for the determination of inhibition constants. Two synthesized inhibitors were shown to exhibit a pronounced selectivity to PlmII (K(i) = 5.5 and 5 nM) in comparison with cathepsin D (K(i) = 230 and 3000 nM, respectively).

Probing the mechanism of purine nucleoside phosphorylase by steady-state kinetic studies and ligand binding characterization determined by fluorimetric titrations

Reversible reaction catalyzed by trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp. with typical and non-typical substrates, including product inhibition patterns of both reaction directions, and interactions of the enzyme with bisubstrate analogue inhibitors, were investigated by the steady-state kinetic methods and fluorimetric titrations. The ligand chromophores exist most probably as neutral species, and not N(1)-H monoanions, in the complex with PNP, as shown by determination of inhibition constants vs. pH. This supports the mechanism in which hydrogen bond interaction of N(1)-H with Glu204 is crucial in the catalytic process. Stoichiometry of ligand binding, with possible exception of hypoxanthine, is three molecules per enzyme trimer. Kinetic experiments show that in principle the Michaelis–Menten model could not properly describe the reaction. However, this model seems to hold for certain experimental conditions. Data presented here are supported by earlier findings obtained by means of fluorimetric titrations and protective effects of ligands on thermal inactivation of the enzyme. All results are consistent with the following mechanism for trimeric PNPs: (i) random binding of substrates, (ii) potent binding and slow release of some reaction products leading to the circumstances that the chemical step is not the slowest one and that rapid-equilibrium assumptions do not hold, (iii) a dual role of phosphate—a substrate and also a reaction modifier.

Molecular modeling and biochemical characterization reveal the mechanism of hepatitis B virus polymerase resistance to lamivudine (3TC) and emtricitabine (FTC).

Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutations on the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitro HBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by determination of inhibition constants (K(i)). Recognition of the natural substrate, dCTP, was assessed by determination of Km values. The results from the in vitro studies were as follows: (i) dCTP substrate binding was largely unaffected by the mutations, with Km changing moderately, only in a range of 0.6 to 2.6-fold; (ii) K(i)s for 3TCTP and FTCTP against Met552Ile/Val mutant HBV polymerases were increased 8- to 30-fold; and (iii) the Leu528Met mutation had a modest effect on direct binding of these beta-L-oxathiolane ring-containing nucleotide analogs. A three-dimensional homology model of the catalytic core of HBV polymerase was constructed via extrapolation from retroviral reverse transcriptase structures. Molecular modeling studies using the HBV polymerase homology model suggested that steric hindrance between the mutant amino acid side chain and lamivudine or emtricitabine could account for the resistance phenotype. Specifically, steric conflict between the Cgamma2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring (common to both beta-L-nucleoside analogs lamivudine and emtricitabine) is proposed to account for the resistance observed upon Met552Ile/Val mutation. The effects of the Leu528Met mutation, which also occurs near the HBV polymerase active site, appeared to be less direct, potentially involving rearrangement of the deoxynucleoside triphosphate-binding pocket residues. These modeling results suggest that nucleotide analogs that are beta-D-enantiomers, that have the sulfur replaced by a smaller atom, or that have modified or acyclic ring systems may retain activity against lamivudine-resistant mutants, consistent with the observed susceptibility of these mutants to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.

New drugs for the Na+/H+ exchanger. Influence of Na+ concentration and determination of inhibition constants with a microphysiometer.

The NHE-1 isoform of the Na+/H+ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensortrade mark microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na+/H+ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na+/H+ antiporter. The Na+/H+ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10-138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a KM = 30 +/- 4 mm for Na+. Addition of either one of the four inhibitors decreased the acidification rate. The IC50 values of the four compounds could be determined as 23 +/- 7 nm for EMD 84021, 5 +/- 1 nm for EMD 94309, 9 +/- 2 nm for EMD 96785 and 8 +/- 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC50 values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na+/H+ antiporter and allows a quantitative kinetic analysis of the proton excretion rate.

The enthalpimetric determination of inhibition constants for the inhibition of urease by fluoride ion

The enthalpimetric determination of inhibition constants for the inhibition of urease by fluoride ion

ACP Broadsheet No 138: May 1993. Gross examination of uterine specimens.

A previous study suggested that fluvoxamine inhibition potency toward CYP1A2 is 10 times greater in vivo than in vitro. The present study was designed to determine whether the same gap exists for CYP2C19, another isozyme inhibited by fluvoxamine. In vitro studies examined the effect of nonspecific binding on the determination of inhibition constant (<i>K</i>_i) values of fluvoxamine toward CYP2C19 in human liver microsomes and in a cDNA-expressed microsomal (Supersomes) system using (<i>S</i>)-mephenytoin as a CYP2C19 probe.<i>K</i>_i values based on total added fluvoxamine concentration (<i>K</i>_i,total) and unbound fluvoxamine concentration (<i>K</i>_i,ub) were calculated, and interindividual variability in<i>K</i>_i values was examined in six nonfatty livers. <i>K</i>_i,total values varied with microsomal protein concentration, whereas the corresponding<i>K</i>_i,ub values were within a narrow range (70–80 nM). In vivo inhibition constants (<i>K</i>_iiv) were obtained from a study of the disposition of a single oral dose (100 mg) of the CYP2C19 probe (<i>S</i>)-mephenytoin in 12 healthy volunteers receiving fluvoxamine at 0, 37.5, 62.6, and 87.5 mg/day to steady state. In this population, the ratio of (<i>S</i>)-4-hydroxy-mephenytoin formation clearances (uninhibited/inhibited) was positively correlated with fluvoxamine average steady-state concentration with an intercept of 0.85 (<i>r</i>^<i>2</i> = 0.88,<i>p</i> &lt; 0.001). The mean (±S.D.) values of<i>K</i>_iiv based on total and unbound plasma concentrations were 13.5 ± 5.6 and 1.9 ± 1.1 nM, respectively. Comparison of in vitro and in vivo<i>K</i>_i values, based on unbound fluvoxamine concentrations, suggests that fluvoxamine inhibition potency is roughly 40 times greater in vivo than in vitro.

Determination of inhibition constants, I50 values and the type of inhibition for enzyme-catalyzed reactions.

A procedure is proposed for determining whether an inhibitor of an enzyme-catalyzed reaction is competitive, noncompetitive, or uncompetitive with respect to the substrate. The method is based on fitting the equation for noncompetitive inhibition to data obtained by measuring the rate of the reaction over a range of substrate and inhibitor concentrations. The results of this fit may suggest that the inhibition may be either competitive or uncompetitive, whereupon the data are reanalyzed using the appropriate equation. Comparison of this second fit with the first using an F test permits a statistical decision to be made on the type of inhibition. The chosen fit yields values and standard errors for the Michaelis-Menten parameters (maximum velocity and Michaelis constant), as well as the inhibition constant(s). From these values it is then possible to predict the I50, and its standard error, at any chosen substrate concentration, thereby facilitating comparison with results obtained with similar inhibitors, for homologous enzymes, or in other laboratories.