Simple Competitive Inhibition Research Articles

Catalytic Differences between Flavohemoglobins of Giardia intestinalis and E. coli.

The sole known heme enzyme of the parasitic protist Giardia intestinalis is a flavohemoglobin (gFlHb) that acts as a nitric oxide dioxygenase (NOD) and protects the organism from the free radical nitric oxide. To learn more about the properties of this enzyme, we measured its nitric oxide dioxygenase, NADH oxidase, and cytochrome c reductase activities and compared these to the activities of the E. coli flavohemoglobin (Hmp). The turnover number for the NOD activity of gFlHb (23 s-1) is about two-thirds of that of Hmp (34 s-1) at pH 6.5 and 37 °C. The two enzymes differ in their sensitivity towards molecules that act as heme ligands. For both gFlHb and Hmp, inhibition with miconazole, a large imidazole ligand, is adequately described by simple competitive inhibition, with KI = 10 μM and 0.27 μM for gFlHb and Hmp, respectively. Inhibition plots with the small ligand imidazole were biphasic, which is consistent with previous experiments with carbon monoxide as a probe that show that the active site of flavohemoglobins exists in two conformations. Interestingly, the largest difference is observed with nitrite, which, like imidazole, also shows a biphasic inhibition plot; however, nitrite inhibits gFlHb at sub-millimolar concentrations while Hmp is not significantly affected. NADH oxidase activity measured under aerobic conditions in the absence of nitric oxide for Hmp was more than twice the activity of gFlHb. The addition of 1 mM hydrogen peroxide in these assays stimulated the NADH oxidase activity of gFlHb but not Hmp. Both enzymes had nearly identical cytochrome c reductase activities but the extent of the contribution of indirect reduction by flavohemoglobin-generated superoxide was much lower with gFlHb (4% SOD-inhibited) than with Hmp (17% SOD-inhibited). Although the active sites of the two enzymes share the same highly conserved residues that are important for catalysis, differences in the distal ligand binding site may account for these differences in activity and sensitivity towards NOD inhibitors. The differences observed in the NADH oxidase and cytochrome c reductase assays suggest that gFlHb may have evolved to protect the protist, which lacks both superoxide dismutase and catalase, from the damaging effects of superoxide by minimizing its production and from peroxide by actively reducing it.

Dissipation kinetics of chlorpyrifos and 3,5,6 trichloro-2-pyridinol under vegetation of different aromatic grasses: Linkage with enzyme kinetics and microbial community of soil

The dissipation of chlorpyrifos (CP) and its hydrolytic metabolite 3,5,6-trichloro-2-pyridinol (TCP) in the soil is crucial for safe agriculture. However, there is still lacking relevant information about its dissipation under different vegetation for remediation purposes. In the present study, evaluation of dissipation of CP and TCP in non-planted and planted soil with different cultivars of three types of aromatic grass viz Cymbopogon martinii (Roxb. Wats), Cymbopogon flexuosus, and Chrysopogon zizaniodes (L.) Nash was examined in light of soil enzyme kinetics, microbial communities, and root exudation. Results revealed that the dissipation of CP was well-fitted into a single first-order exponential model (SFO). A significant reduction in the half-life (DT50) of CP was observed in planted soil (30–63 days) than in non-planted soil (95 days). The presence of TCP in all soil samples was observed. The three types of the inhibitory effect of CP i.e. linear mixed inhibition (increase in enzyme-substrate affinity (Km) and decrease in enzyme pool (Vmax), un-competitive inhibition (decrease in Km and Vmax), and simple competitive inhibition were observed on soil enzymes involved in mineralization of carbon, nitrogen, phosphorus, and sulfur. The improvement in the enzyme pool (Vmax) was observed in planted soil. Streptomyces, Clostridium, Kaistobacter, Planctomyces, and Bacillus were the dominant genera in CP stress soil. CP contamination in soil demonstrated a reduction of richness in microbial diversity and enhancement of functional gene family related to cellular process, metabolism, genetic, and environmental information processing. Among all the cultivars, C. flexuosus cultivars demonstrated a higher dissipation rate of CP along with more root exudation.

Erythrocyte-Specific Antibodies As a Potential New Therapeutic Modality in Autoimmune and Inflammatory Disorders Beyond Immune Thrombocytopenia (ITP)

ITP is an autoimmune bleeding disorder characterized by platelet destruction and suppression of platelet production. In the majority of patients, platelet destruction due to IgG antibodies reactive with platelet GPIIbIIIa results in platelet phagocytosis by phagocytic cells in the mononuclear phagocytic system. Anti-D is an effective first-line therapy for patients with ITP and was originally brought into practice based on the ability of anti-D to competitively inhibit opsonized-platelet clearance by phagocytic cells. Similar to anti-D, an RBC-specific monoclonal antibody (TER-119) is also able to inhibit platelet clearance in a murine model of passive ITP, also ostensibly due to RBC clearance and competitive inhibition of phagocytic cells. Here, we show that TER-119 only caused measurable anemia after 3 hours post-injection while its ability to ameliorate thrombocytopenia in the passive-murine ITP model was clearly evident by 30 minutes post-injection. Further, TER-119 mediated maximal anemia 4 days after its injection, yet displayed no measurable ameliorative effects in passive-ITP at this time point. Based on these observations, we hypothesized that anti-inflammatory activity, rather than simple competitive inhibition of opsonized-platelet clearance, might be involved in this mechanism. To test this hypothesis, the ability of TER-119 to successfully treat inflammatory diseases not involving antibody-mediated splenic cellular sequestration were examined. In the K/BxN serum-transfer model of arthritis, recipient C57BL/6 mice are injected with K/BxN serum and a rapidly progressing arthritis occurs due to antibodies specific to the self-protein glucose-6-phosphate isomerase. Using this model, the same dose of TER-119 (50 μg/mouse) which was effective in murine ITP could significantly prevent the induction of inflammatory arthritis. In separate experiments, mice were allowed to develop significant arthritis for 5 days and then injected with TER-119. These arthritic mice underwent a reversal in disease severity with significant and substantial decreases in joint swelling and clinical score evident 24 hours after injection of TER-119. In fact three days after injection with TER-119, clinical scores and ankle width measurements returned to baseline. The function of the Fc region was important in the anti-inflammatory activity of TER-119 as deglycosylation of the Fc region of the antibody (known to inhibit IgG Fc region effector functions) dramatically reduced anti-inflammatory function in the arthritis model. To determine if TER-119 could also ameliorate disease activity in an unrelated (non-autoimmune) inflammatory disease model, TER-119 was also examined in a murine model of transfusion-related acute lung injury (TRALI). TRALI is a syndrome of respiratory distress primarily triggered by leukocyte-reactive antibodies from blood product transfusions and is the leading cause of transfusion-related mortality. TRALI was induced by injecting mice with the anti-MHC class I antibody (34-1-2s) which induced a rapid decrease in body temperature and induced significant lung damage, assessed by the presence of pulmonary edema. Mice pretreated with TER-119 (50 μg) underwent a rescue from hypothermia and exhibited an almost complete inhibition of lung edema. We conclude that TER-119 has significant anti-inflammatory activity and suggest that RBC specific antibodies such as anti-D may have significant therapeutic potential in inflammatory disorders beyond just ITP. DisclosuresSemple:Novartis: Consultancy; Rigel: Consultancy; Amgen: Consultancy; UCB: Consultancy. Lazarus:CSL pharmaceuticals: Research Funding; Momenta pharmaceuticals: Honoraria; Rigel pharmaceuticals: Research Funding; St.Michael's Hosp/The Canadian Blood Services: Other: Patents on monoclonal antibodies as IVIg and anti-D replacements.

Decreased behavioural and neurochemical effects of angiotensin IV following prenatal alcohol exposure in the mouse

Angiotensin IV (ang IV) is known to improve learning and memory in animal models but the mechanism is unclear. We have previously demonstrated sex differences in the pro-cognitive effects of ang IV, and that prenatal alcohol exposure (PAE) abolishes these effects. This study aimed to explore a possible mechanism underlying the sex differences and the effects of PAE in male mice.Mouse breeding harems received 5% ethanol in drinking water throughout pregnancy and lactation in a two-bottle schedule. The effects of ang IV were assessed in offspring at 4 months of age using the open field test, novel object recognition test and elevated plus maze. Aminopeptidase activity of brain insulin-regulated aminopeptidase (IRAP), a putative target of ang IV, was determined.As seen in a previous similar study, ang IV administered immediately after the second training trial significantly improved novel object recognition 24 h later in male mice but not female. PAE abolished this pro-cognitive effect in males. PAE also increased anxiety-like behaviour in male but not female offspring. Ang IV decreased the aminopeptidase activity of brain IRAP in control male, but not female, mice; PAE abolished this inhibitory effect.Ang IV improved memory consolidation in male but not female mice and PAE abolished this effect in the males. While the effects of PAE may be related to increased anxiety; ang IV decreased the aminopeptidase activity in male but not female mice and PAE abolished this inhibitory effect. The results therefore suggest that improvements in learning and memory induced by peripheral administration of ang IV correlate with a reduction of the enzyme activity of IRAP. This is the first demonstration that ang IV administered peripherally can induce long-term (24 h) changes in IRAP function which are probably not simple competitive inhibition and the first demonstration that PAE alters IRAP activity.

Two‐Dimensional Crowding Uncovers a Hidden Conformation of α‐Synuclein

AbstractThe intrinsically disordered protein (IDP), α‐synuclein (αS), is well‐known for phospholipid membrane binding‐coupled folding into tunable helical conformers. Here, using single‐molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction–folding landscape of αS can be tuned by two‐dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS–membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher‐affinity module remains membrane‐bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane‐binding IDPs with multiple binding modes.

Two-Dimensional Crowding Uncovers a Hidden Conformation of α-Synuclein.

The intrinsically disordered protein (IDP), α-synuclein (αS), is well-known for phospholipid membrane binding-coupled folding into tunable helical conformers. Here, using single-molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction-folding landscape of αS can be tuned by two-dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS-membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher-affinity module remains membrane-bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane-binding IDPs with multiple binding modes.

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10Å from the active site, while epitope β is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a β epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.

The mechanism of the inhibitory action of salicylate on glutamyl-transfer ribonucleic acid synthetase in vitro.

Abstract Salicylate inhibits glutamyl-transfer ribonucleic acid synthetase by simple competitive inhibition with respect to glutamate and mixed competitive-non-competitive inhibitions with ATP and tRNA. It is suggested that competition between salicylate and amino-acid explains the variable inhibition of the synthetases by the drugs in vitro. The action of salicylate is consistent with a mechanism for glutamyl-tRNA formation involving a concerted interaction of all three substrates. The drug may selectively interfere with protein synthesis in vivo.

The effect of DSP-4 (N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine) on monoamine oxidase activities in tissues of the rat.

The in vitro inhibition of monoamine oxidase (MAO) in rat liver, and of the clorgyline-resistant amine oxidase (CRAO) in rat heart and aorta, by DSP-4 (N-[2-chloroethyl]-N-ethyl-2-bromobenzylamine) has been studied. Inhibition of each enzyme activity was independent of prolonged preincubation, was reversed by dialysis, and also Ackermann-Potter plots were consistent with reversible inhibition. Simple linear competitive inhibition of MAO-A and MAO-B was observed, with Ki values of 6 x 10(-6) and 8 x 10(-5) M, respectively. CRAO was inhibited in a mixed, non-competitive manner (Ki of 3.2 x 10(-5) and 7.8 x 10(-6) M in heart and aorta, respectively) which conformed to a kinetic model in which the binding of DSP-4 to CRAO increased the affinity of substrate binding, but prevented product formation. The possible significance of these results for the in vivo actions of the drug is discussed.

Evidence for noncompetitive modulation of substrate‐induced serotonin release

Prior work indicated that serotonin transporter (SERT) inhibitors competitively inhibit substrate-induced [(3)H]5-HT release, producing rightward shifts in the substrate-dose response curve and increasing the EC(50) value without altering the E(max). We hypothesized that this finding would not generalize across a number of SERT inhibitors and substrates, and that the functional dissociation constant (Ke) of a given SERT inhibitor would not be the same for all tested substrates. To test this hypothesis, we utilized a well-characterized [(3)H]5-HT release assay that measures the ability of a SERT substrate to release preloaded [(3)H]5-HT from rat brain synaptosomes. Dose-response curves were generated for six substrates (PAL-287 [naphthylisopropylamine], (+)-fenfluramine, (+)-norfenfluramine, mCPP [meta-chlorophenylpiperazine], (±)-MDMA, 5-HT) in the absence and presence of a fixed concentration of three SERT inhibitors (indatraline, BW723C86, EG-1-149 [4-(2-(benzhydryloxy)ethyl)-1-(4-bromobenzyl)piperidine oxalate]). Consistent with simple competitive inhibition, all SERT inhibitors increased the EC(50) value of all substrates. However, in many cases a SERT inhibitor decreased the E(max) value as well, indicating that in the presence of the SERT inhibitor the substrate became a partial releaser. Moreover, the Ke values of a given SERT inhibitor differed among the six SERT substrates, indicating that each inhibitor/substrate combination had a unique interaction with the transporter. Viewed collectively, these findings suggest that it may be possible to design SERT inhibitors that differentially regulate SERT function.

Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of Protease Inhibitors

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a bifunctional enzyme with a protease and a helicase functionality located in each of the two domains of the single peptide chain. There is little experimental evidence for a functional role of this unexpected arrangement since artificial single domain forms of both enzymes are catalytically competent. We have observed that low concentrations of certain protease inhibitors activate the protease of full-length NS3 from HCV genotype 1a with up to 100%, depending on the preincubation time and the inhibitor used. The activation was reduced, but not eliminated, by increased ionic strength, lowered glycerol concentration, or lowered pH. In all cases, it was at the expense of a significant loss of activity. Activation was not seen with the artificial protease domain of genotype 1b NS3 fused with a fragment of the NS4A cofactor. This truncated and covalently modified enzyme form was much less active and exhibited fundamentally different catalytic properties to the full-length NS3 protease without the fused cofactor. The most plausible explanation for the activation was found to involve a slow transition between two enzyme conformations, which differed in their catalytic ability and affinity for inhibitors. Equations derived based on this assumption resulted in better fits to the experimental data than the equation for simple competitive inhibition. The mechanism may involve an inhibitor-induced stabilization of the helicase domain in a conformation that enhances the protease activity, or an improved alignment of the catalytic triad in the protease. The proposed mnemonic mechanism and derived equations are viable for both these explanations and can serve as a basic framework for future studies of enzymes activated by inhibitors or other ligands.

Tyrosinase Inhibitory Polyphenols from Roots ofMorus lhou

Twelve polyphenols (1-12) possessing tyrosinase inhibitory properties were isolated from the methanol (95%) extract of Morus lhou. The isolated compounds consisted of four flavanones (1-4), four flavones (5-8), and four phenylbenzofuranes (9-12). Moracin derivative 12 proved to be new a compound which was fully characterized. Compounds 1-12 were evaluated for both monophenolase and diphenolase (the two steps catalyzed by tyrosinase) inhibition to identify the structural characteristics required for mushroom tyrosinase inhibition. We observed that all parent compounds (1, 5, and 9) possessing an unsubstituted resorcinol group were highly effective inhibitors of monophenolase activity (IC(50) values of 1.3, 1.2, and 7.4 microM). The potency of the inhibitors diminished with alkyl substitution on either the aromatic ring or the hydroxyl functions. Interestingly, flavone 5 was shown to possess only monophenolase inhibitory activity, but flavanone 1 and phenylbenzofuran 9 inhibited diphenolase as well as monophenolase significantly. The inhibitory mode of these species was also dependent upon the skeleton: phenylbenzofuran 9 manifested a simple competitive inhibition mode for monophenolase and diphenolase; on the other hand flavanone 1 (monophenolase, k(3) = 0.1966 min(-1) microM(-1), k(4) = 0.0082 min(-1), and K(i)(app) = 0.0468 microM; diphenolase, k(3) = 0.0014 min(-1) microM(-1), k(4) = 0.0013 min(-1), and K(i)(app) = 0.8996 microM) and flavone 5 both showed time-dependent inhibition against monophenolase. Compound 1 operated according to the simple reversible slow binding model whereas compound 5 operated under the enzyme isomerization model.

Characterizing metabolic inhibition using electrochemical enzyme/DNA biosensors.

Studies of metabolic enzyme inhibition are necessary in drug development and toxicity investigations as potential tools to limit or prevent appearance of deleterious metabolites formed, for example, by cytochrome (cyt) P450 enzymes. In this paper, we evaluate the use of enzyme/DNA toxicity biosensors as tools to investigate enzyme inhibition. We have examined DNA damage due to cyt P450cam metabolism of styrene using DNA/enzyme films on pyrolytic graphite (PG) electrodes monitored via Ru(bpy)(3)(2+)-mediated DNA oxidation. Styrene metabolism initiated by hydrogen peroxide was evaluated with and without the inhibitors, imidazole, imidazole-4-acetic acid, and sulconazole (in micromolar range) to monitor DNA damage inhibition. The initial rates of DNA damage decreased with increased inhibitor concentrations. Linear and nonlinear fits of Michaelis-Menten inhibition models were used to determine apparent inhibition constants (K(I)*) for the inhibitors. Elucidation of the best fitting inhibition model was achieved by comparing correlation coefficients and the sum of the square of the errors (SSE) from each inhibition model. Results confirmed the utility of the enzyme/DNA biosensor for metabolic inhibition studies. A simple competitive inhibition model best approximated the data for imidazole, imidazole-4-acetic acid and sulconazole with K(I)* of 268.2, 142.3, and 204.2 microM, respectively.

A Combined Model for Predicting CYP3A4 Clinical Net Drug-Drug Interaction Based on CYP3A4 Inhibition, Inactivation, and Induction Determined in Vitro

Although approaches to the prediction of drug-drug interactions (DDIs) arising via time-dependent inactivation have recently been developed, such approaches do not account for simple competitive inhibition or induction. Accordingly, these approaches do not provide accurate predictions of DDIs arising from simple competitive inhibition (e.g., ketoconazole) or induction of cytochromes P450 (e.g., phenytoin). In addition, methods that focus upon a single interaction mechanism are likely to yield misleading predictions in the face of mixed mechanisms (e.g., ritonavir). As such, we have developed a more comprehensive mathematical model that accounts for the simultaneous influences of competitive inhibition, time-dependent inactivation, and induction of CYP3A in both the liver and intestine to provide a net drug-drug interaction prediction in terms of area under the concentration-time curve ratio. This model provides a framework by which readily obtained in vitro values for competitive inhibition, time-dependent inactivation and induction for the precipitant compound as well as literature values for f(m) and F(G) for the object drug can be used to provide quantitative predictions of DDIs. Using this model, DDIs arising via inactivation (e.g., erythromycin) continue to be well predicted, whereas those arising via competitive inhibition (e.g., ketoconazole), induction (e.g., phenytoin), and mixed mechanisms (e.g., ritonavir) are also predicted within the ranges reported in the clinic. This comprehensive model quantitatively predicts clinical observations with reasonable accuracy and can be a valuable tool to evaluate candidate drugs and rationalize clinical DDIs.

A Novel Fluorescence Intensity Screening Assay Identifies New Low-Molecular-Weight Inhibitors of the gp41 Coiled-Coil Domain of Human Immunodeficiency Virus Type 1

A metallopeptide-based fluorescence assay has been designed for the detection of small-molecule inhibitors of human immunodeficiency virus type 1 gp41, the viral protein involved in membrane fusion. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer C-terminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The two peptides span a hydrophobic pocket previously defined in the literature. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorophore-labeled HR2 peptide to be followed by fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and we show that they can be detected in the assay from an increase in the fluorescence that is correlated with the potency of the compound. Assay optimization and validation have resulted in a simple quantitative competitive inhibition assay for fusion inhibitors that bind in the hydrophobic pocket. The assay has an assay quality factor (Z') of 0.88 and can rank order inhibitors at 10 microM concentration with K(i)s in the range of 0.2 microM to 30 microM, an ideal range for drug discovery. Screening of a small peptidomimetic library has yielded three new low-molecular-weight gp41 inhibitors. In vitro syncytium inhibition assays confirmed that the compounds inhibited cell-cell fusion in the low micromolar range. These lead compounds provide a new molecular scaffold for the development of fusion inhibitors.

Potent Mechanism-based Inhibitors for Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k(on) of 10(2) to 10(4) M(-1) s(-1) and the reversal of the process is slow (k(off) of 10(-3) to 10(-4) s(-1)). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.

Effect of desferrioxamine and metals on the hydroxylases in the oxygen sensing pathway

Hypoxia-inducible transcription factor (HIF) is regulated by two oxygen-dependent events that are catalyzed by the HIF prolyl 4-hydroxylases (HIF-P4Hs) and HIF asparaginyl hydroxylase (FIH). We have purified the three recombinant human HIF-P4Hs to near homogeneity and characterized their catalytic properties and inhibition and those of FIH. The specific activities of the HIF-P4Hs were at least 40-50 mol/mol/min, and they and FIH catalyzed an uncoupled decarboxylation of 2-oxoglutarate in the absence of any peptide substrate. The purified HIF-P4Hs showed considerable activities even without added Fe2+, their apparent Km values for iron being markedly lower than that of FIH. Desferrioxamine and several metals were effective inhibitors of FIH, but surprisingly, ineffective inhibitors of the HIF-P4Hs in vitro, especially of HIF-P4H-2. Desferrioxamine and cobalt were more effective in cultured insect cells synthesizing recombinant HIF-P4H-2, but complete inhibition was not achieved and most of the enzyme was inactivated irreversibly. Cobalt also rapidly inactivated HIF-P4Hs during storage at 4 degrees C. The well-known stabilization of HIF-alpha by cobalt and nickel is thus not due to a simple competitive inhibition of HIF-P4Hs. The effective inhibition of FIH by these metals and zinc probably leads to full transcriptional activity of HIF-alpha even in concentrations that produce no stabilization of HIF-alpha.

The effect of valproic acid on drug and steroid glucuronidation by expressed human UDP-glucuronosyltransferases

Valproic acid glucuronidation kinetics were carried out with three human UGT isoforms: UGT1A6, UGT1A9, and UGT2B7 as well as human liver and kidney microsomes. The glucuronidation of valproic acid was typified by high K m values with microsomes and expressed UGTs (2.3–5.2 mM). The ability of valproic acid to interact with the glucuronidation of drugs, steroids and xenobiotics in vitro was investigated using the three UGT isoforms known to glucuronidate valproic acid. In addition to this the effect of valproic acid was investigated using two other UGT isoforms: UGT1A1 and UGT2B15 which do not glucuronidate valproic acid. Valproic acid inhibited UGT1A9 catalyzed propofol glucuronidation in an uncompetitive manner and UGT2B7 catalyzed AZT glucuronidation competitively ( K i =1.6±0.06 mM). Valproate significantly inhibited UGT2B15 catalyzed steroid and xenobiotic glucuronidation although valproate was not a substrate for this UGT isoform. No significant inhibition of UGT1A1 or UGT1A6 by valproic acid was observed. These data indicate that valproic acid inhibition of glucuronidation reactions is not always due to simple competitive inhibition of substrates.

Effects of proline analog binding on the spectroscopic and redox properties of PutA.

The PutA flavoprotein regulates proline metabolism in Escherichia coli by performing two distinct functions. First, in the cytoplasm, PutA represses transcription of the put (proline utilization) regulon. Second, PutA associates with the membrane to oxidize proline to glutamate using discrete proline dehydrogenase and Delta(1)-pyrroline-5-carboxylate dehydrogenase domains. Here, we identify a proline analog that will be useful for testing the role substrate binding has in regulating PutA functions. L-Tetrahydro-2-furoic acid (L-THFA) was found to display simple competitive inhibition of proline dehydrogenase activity in PutA (apparent K(i)=0.2mM) and to perturb the flavin adenine dinucleotide (FAD) absorbance spectrum upon complexation to PutA. At pH 7.5, a reduction potential (E(m)) of -0.089V for the FAD/FADH(2) couple in L-THFA-complexed PutA was determined by potentiometric titrations. The E(m) value for L-THFA-complexed PutA is 12mV more negative than the E(m) for uncomplexed PutA (E(m)=-0.077V, pH 7.5) and corresponds to just a twofold increase in the dissociation constant of L-THFA with PutA upon reduction of FAD.

Substrate−Inhibitor Interactions in the Kinetics of α-Amylase Inhibition by Ragi α-amylase/Trypsin Inhibitor (RATI) and Its Various N-Terminal Fragments

The ragi alpha-amylase/trypsin bifunctional inhibitor (RATI) from Indian finger millet, Ragi (Eleucine coracana Gaertneri), represents a new class of cereal inhibitor family. It exhibits a completely new motif of trypsin inhibitory site and is not found in any known trypsin inhibitor structures. The alpha-amylase inhibitory site resides at the N-terminal region. These two sites are independent of each other and the inhibitor forms a ternary (1:1:1) complex with trypsin and alpha-amylase. The trypsin inhibition follows a simple competitive inhibition obeying the canonical serine protease inhibitor mechanism. However, the alpha-amylase inhibition kinetics is a complex one if larger (> or =7 glucose units) substrate is used. While a complete inhibition of trypsin activity can be achieved, the inhibition of amylase is not complete even at very high molar concentration. We have isolated the N-terminal fragment (10 amino acids long) by CNBr hydrolysis of RATI. This fragment shows a simple competitive inhibition of alpha-amylase activity. We have also synthesized various peptides homologous to the N-terminal sequence of RATI. These peptides also show a normal competitive inhibition of alpha-amylase with varying potencies. It has also been shown that RATI binds to the larger substrates of alpha-amylase. In light of these observations, we have reexamined the binding of proteinaceous inhibitors to alpha-amylase and its implications on the mechanism and kinetics of inhibition.