Spontaneous epimerization of (S)-deoxycoformycin and interaction of (R)-deoxycoformycin, (S)-deoxycoformycin, and 8-ketodeoxycoformycin with adenosine deaminase

(R)-Deoxycoformycin (pentostatin), (S)-deoxycoformycin, and 8-ketodeoxycoformycin were compared as inhibitors of calf intestine adenosine deaminase. In contrast to (R)-deoxycoformycin, which had been demonstrated as a tight-binding inhibitor with a dissociation constant of 2.5 X 10(-12) M [Agarwal, R. P., Spector, T., & Parks, R. E., Jr. (1977) Biochem. Pharmacol. 26, 359-367], (S)-deoxycoformycin and 8-ketodeoxycoformycin are slope-linear competitive inhibitors with respect to adenosine. The kinetic constants are 33 microM for inhibition by (S)-deoxycoformycin, 43 microM for 8-ketodeoxycoformycin, and 16 microM for the Km for adenosine. The stereochemistry of carbon 8 of the diazepine ring therefore causes a (1.3 X 10(7]-fold change in the affinity for the enzyme which is specific for the R configuration. This difference is attributed to an induced conformational change which cannot be initiated by the S isomer or the 8-keto analogue of (R)-deoxycoformycin. The studies were complicated by the need to remove traces of tight-binding inhibitor(s) from (S)-deoxycoformycin, since as little as 0.001% of the R isomer causes significant inhibition. The R and S isomers of deoxycoformycin are unstable in neutral or mildly acidic aqueous solutions. Isomerization of the secondary hydroxyl at carbon 8 of the diazepine ring is one of the reactions, resulting in S to R and R to S conversions for deoxycoformycins. Opening of the aglycon is also a major reaction. The tight-binding inhibitor generated from (S)-deoxycoformycin was identified as (R)-deoxycoformycin by high-pressure liquid chromatography, spectroscopy, circular dichroism, and chemical criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological functions of serine proteases in mast cells in allergic inflammation

Serine proteases in mast cell granules, such as chymase, atypical chymase, and tryptase, which are major proteins in the granules, may play important roles in the process of immunoglobulin E (IgE)-mediated degranulation and in pathobiological alterations in tissues. Indeed, inhibitors of chymase, substrate analogs, and antichymase F(ab')2, but not inhibitors of tryptase, markedly inhibited histamine release induced by IgE-receptor bridging but not that induced by Ca ionophore. In contrast, inhibitors of metalloprotease inhibited histamine release induced not only by IgE-receptor bridging but also by Ca ionophore. These results suggest that chymase and metalloprotease are involved at different steps in the process of degranulation. The extents of inhibition of histamine release were closely correlated with the amounts of the inhibitors of chymase accumulated in the granules. After degranulation, the released proteases may in part contribute to pathobiological alterations in allergic disorders through generations of C3a anaphylatoxin and thrombin by human and rat tryptase, respectively, and those of angiotensin II and a chemotactic factor of neutrophils by human and rat chymase, respectively. Moreover, chymase and atypical chymase from rat were shown to destroy type IV collagen, and human tryptase was found to hydrolyze various plasma proteins, such as fibrinogen and high-molecular-weight kininogen. The biological activities of tryptase and chymase from rat may be regulated by their dissociation from and association with trypstatin, an endogenous inhibitor of these proteases.     

Purification and preliminary characterization of a macromolecular inhibitor of glucocorticoid receptor binding to DNA

Rat liver cytosol contains a heat-labile macromolecule that inhibits the binding of the transformed glucocorticoid-receptor complex to nuclei or DNA-cellulose (Milgrom, E., and Atger, M. (1975) J. Steroid Biochem. 6, 487-492; Simons, S. S., Jr., Martinez, H. M., Garcea, R. L., Baxter, J. D., and Tomkins, G. M. (1976) J. Biol. Chem. 251, 334-343. We have developed a quantitative assay for the inhibitor and have purified it 600-700-fold by ammonium sulfate precipitation, ethanol precipitation, and phosphocellulose and Sephacryl S-300 chromatography. The inhibitory activity copurifies with a Mr = 37,000 protein doublet. Under low salt conditions, both the inhibitory activity and the 37-kDa protein doublet behave as high Mr aggregates that subsequently dissociate in the presence of salt. The inhibitor is positively charged at physiological pH, and it is not affected by digestion with several serine proteases or RNase. The inhibitor does not affect the transformation process, and it does not cause the release of steroid-receptor complexes that have been prebound to DNA-cellulose. The inhibitor preparation does not cleave receptors in L-cell cytosol that are covalently labeled with the site-specific affinity steroid [3H]dexamethasone 21-mesylate. If the steroid-receptor complex is first separated from the great majority of cytosol protein by transforming it and binding it to DNA-cellulose, addition of the inhibitor preparation results in receptor cleavage. Under these conditions, cleavage can be blocked with 1-chloro-3-tosylamido-7-amino-L-2-heptanone and antipain, but protease inhibitors do not affect the inhibition of DNA binding that occurs in whole cytosol. The inhibitor acts through an interaction with the receptor, not with DNA. We suggest that the inhibitor may prove to be a useful tool for studying the interaction of the steroid-receptor complex with DNA or nuclei and speculate that it may be important in determining normal events of the receptor cycle as they occur in the intact cell.     

Tryptase in rat mast cells: properties and inhibition by various inhibitors in comparison with chymase

Previous studies with trans-4-(guanidinomethyl)cyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut), a trypsin inhibitor, strongly suggested the involvement of a trypsin-like protease in histamine release from mast cells induced by various secretagogues (Takei, M., Matumoto, T., Endo, K. & Muramatu, M. (1988) Agents and Actions, in press; Takei, M., Matumoto, T., Ito, T., Endo, K. & Muramatu, M.; Takei, M., Matumoto, T., Endo, K. & Muramatu, M. and Takei, M., Matumoto, T., Urashima, H., Endo, K. & Muramatu, M., unpublished results). Two serine proteases, chymase (Benditt, E.F. & Arase, M. (1959) J. Exp. Med. 110, 451-460) and tryptase Kido, H., Fukusen, N. & Katunuma, N. (1985) Arch. Biochem. Biophys. 239, 436-443) were demonstrated in rat peritoneal mast cells. Both enzymes were purified and the effects of inhibitors for trypsin and chymotrypsin on these proteases were examined. The trypsin-like protease was found in saline extract and purified by successive chromatographies on Sephadex G-100 and DEAE-cellulose columns. The molecular mass of this protease was apparently 120,000 Da. This protease showed maximal activity at pH 7.1 and was named pH 7 tryptase. Chymase was obtained from 1.5M NaCl extract. pH 7 Tryptase markedly hydrolysed Boc-Phe-Ser-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec among the various substrates containing arginyl and lysyl bonds but did not cleave Tos-Arg-OMe. Tos-Lys-CH2Cl and diisopropylfluorophosphate strongly inhibited this protease. Various inhibitors for trypsin inhibited pH 7 tryptase, and those for chymotrypsin inhibited chymase. Among the esters of GMCHA examined, GMCHA-OPhBut most strongly and competitively inhibited pH 7 tryptase but it had no effect on chymase.     

Linear relationships between the ligand binding energy and the activation energy of time-dependent inhibition of steroid 5alpha-reductase by delta 1-4-azasteroids

The inhibition of steroid 5alpha-reductase (5AR) by Delta(1)-4-azasteroids is characterized by a two-step time-dependent kinetic mechanism where inhibitor combines with enzyme in a fast equilibrium, defined by the inhibition constant K(i), to form an initial reversible enzyme-inhibitor complex, which subsequently undergoes a time-dependent chemical rearrangement, defined by the rate constant k(3), leading to the formation of an apparently irreversible, tight-binding enzyme-inhibitor complex (Tian, G., Mook, R. A., Jr., Moss, M. L., and Frye, S. V. (1995) Biochemistry 34, 13453-13459). A detailed kinetic analysis of this process with a series of Delta(1)-4-azasteroids having different C-17 substituents was performed to understand the relationships between the rate of time-dependent inhibition and the affinity of the time-dependent inhibitors for the enzyme. A linear correlation was observed between ln(1/K(i)), which is proportional to the ligand binding energy for the formation of the enzyme-inhibitor complex, and ln(1/(k(3)/K(i))), which is proportional to the activation energy for the inhibition reaction under the second order reaction condition, which leads to the formation of the irreversible, tight-binding enzyme-inhibitor complex. The coefficient of the correlation was -0.88 +/- 0.07 for type 1 5AR and -1.0 +/- 0.2 for type 2 5AR. In comparison, there was no obvious correlation between ln(1/K(i)) and ln(1/k(3)), which is proportional to the activation energy of the second, time-dependent step of the inhibition reaction. These data are consistent with a model where ligand binding energies provided at C-17 of Delta(1)-4-azasteroids is fully expressed to lower the activation energy of k(3)/K(i) with little perturbation of the energy barrier of the second, time-dependent step.     

Synthesis and evaluation of 4-substituted benzylamine derivatives as beta-tryptase inhibitors

Since beta-tryptase is considered a critical mediator of asthma, potent tryptase inhibitors may be useful as new agents for the treatment of asthma. We investigated 4-substituted benzylamine derivatives and obtained M58539 (15h) as a potent inhibitor of beta-tryptase (IC50 = 5.0 nM) with high selectivity against other serine proteases, low molecular weight, clog P value less than 5, lack of amidino and guanidino groups, and independence of Zn2+ ion.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

Recruiting Zn2+ to mediate potent, specific inhibition of serine proteases.

As regulators of ubiquitous biological processes, serine proteases can cause disease states when inappropriately expressed or regulated, and are thus rational targets for inhibition by drugs. Recently we described a new inhibition mechanism applicable for the development of potent, selective small molecule serine protease inhibitors that recruit physiological Zn2+ to mediate high affinity (sub-nanomolar) binding. To demonstrate some of the structural principles by which the selectivity of Zn2+-mediated serine protease inhibitors can be developed toward or against a particular target, here we determine and describe the structures of thrombin-BABIM-Zn2+, -keto-BABIM-Zn2+, and -hemi-BABIM-Zn2+ (where BABIM is bis(5-amidino-2-benzimidazolyl)methane, keto-BABIM is bis(5-amidino-2-benzimidazolyl)methane ketone, and hemi-BABIM is (5-amidino-2-benzimidazolyl)(2-benzimidazolyl)methane), and compare them with the corresponding trypsin-inhibitor-Zn2+ complexes. Inhibitor binding is mediated by a Zn ion tetrahedrally coordinated by two benzimidazole nitrogen atoms of the inhibitor, by N(epsilon2)His57, and by O(gamma)Ser195. The structures of Zn2+-free trypsin-BABIM and -hemi-BABIM were also determined at selected pH values for comparison with the corresponding Zn2+-mediated complexes. To assess some of the physiological parameters important for harnessing Zn2+ as a co-inhibitor, crystal structures at multiple pH and [Zn2+] values were determined for trypsin-keto-BABIM. The Kdvalue of Zn2+ for the binary trypsin-keto-BABIM complex was estimated to be <12 nM at pH 7.06 by crystallographic determination of the occupancy of bound Zn2+ in trypsin-keto-BABIM crystals soaked at this pH in synthetic mother liquor containing inhibitor and 100 nM Zn2+. In synthetic mother liquor saturated in Zn2+, trypsin-bound keto-BABIM is unhydrated at pH 9.00 and 9.93, and has an sp2 hybridized ketone carbon bridging the 5-amidinobenzimidazoles, whereas at pH 7.00 and 8.00 it undergoes hydration and a change in geometry upon addition of water to the bridging carbonyl group. To show how Zn2+ could be recruited as a co-inhibitor of other enzymes, a method was developed for locating in protein crystals Zn2+ binding sites where design of Zn2+-mediated ligands can be attempted. Thus, by soaking trypsin crystals in high concentrations of Zn2+ in the absence of a molecular inhibitor, the site where Zn2+ mediates binding of BABIM and analogs was identified, as well as another Zn2+ binding site.     

Synthesis of potent and selective 2-azepanone inhibitors of human tryptase

The serine protease tryptase has been associated with a broad range of allergic and inflammatory diseases and, in particular, has been implicated as a critical mediator of asthma. The inhibition of tryptase therefore has the potential to be a valuable therapy for asthma. The synthesis, employing solution phase parallel methods, and SAR of a series of novel 2-azepanone tryptase inhibitors are presented. A member of this series, 8t, was identified as a potent inhibitor of human tryptase (IC(50)=38 nM) with selectivity >/=330-fold versus related serine proteases (trypsin, plasmin, uPA, tPA, APC, alpha-thrombin, and FXa) [corrected].     

Thrombin Inhibitors as Antithrombotic Agents: The Importance of Rapid Inhibition

Abstract

For use as an antithrombotic agent, a thrombin inhibitor must be potent and specific, i.e., it should not significantly inhibit the proteases of the anticoagulation (activated protein C) and fibrinolytic systems (plasminogen activator and plasmin). Previous evaluation of potency and specificity has been based on inhibition constants (K_i values). However, consideration of the kinetic parameters for natural plasma serine protease inhibitors indicates that a low K_i value with thrombin is not sufficient; the inhibited complex must also form rapidly. Moreover, potent inhibition of activated protein C and plasmin could be tolerated providing the inhibited complex only forms slowly. An ideal profile of kinetic parameters with thrombin, activated protein C and plasmin is formulated and discussed in relation to various classes of thrombin inhibitors. Examination of kinetic data for thrombin inhibitors currently in clinical trials (hirudin and hirulog) indicates that they possess this ideal profile of kinetic parameters.     

Multiple inhibitor analysis of the brequinar and leflunomide binding sites on human dihydroorotate dehydrogenase

Brequinar and the active metabolite of leflunomide, A77 1726, have been clearly shown to inhibit human dihydroorotate dehydrogenase (DHODH), but conflicting mechanisms for their inhibition have been reported. DHODH catalyses the conversion of dihydroorotate (DHO) to orotate concurrent with the reduction of ubiquinone. This study presents data that indicates brequinar is a competitive inhibitor versus ubiquinone; A77 1726 is noncompetitive versus ubiquinone and both are uncompetitive versus DHO. 2-Phenyl 5-quinolinecarboxylic acid (PQC), the core moiety of brequinar also shows competitive inhibition versus ubiquinone. Multiple inhibition experiments indicate that PQC (and thus brequinar) and A77 1726 have overlapping binding sites. Both PQC and A77 1726 are also mutually exclusive with barbituric acid (a competitive inhibitor versus DHO). In addition, we failed to observe brequinar binding to E.orotate by isothermal titration calorimetry (ITC). These results indicate that the E.DHO.inhibitor and E.orotate.inhibitor ternary complexes do not form. The absence of these complexes is consistent with the two-site ping-pong mechanism reported for DHODH. This kinetic data suggests that recent crystal structures of human DHODH complexed with orotate and A77 1726 or brequinar may not represent the relevant physiological binding sites for these inhibitors [Liu, S., Neidhardt, E. A., Grossman, T. H., Ocain, T., and Clardy J. (2000) Structure 8, 25-33].     

Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase.

Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the alpha-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn(2+) (K(i) approximately 0.15 microm) in both directions. Steady-state kinetic studies revealed that Zn(2+) competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn(2+) with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn(2+) accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 microm. Activation is a consequence of Zn(2+) binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn(2+) on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn(2+) can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.     

A potent oligosaccharyl transferase inhibitor that crosses the intracellular endoplasmic reticulum membrane

Recent work has resulted in the development of potent inhibitors of oligosaccharyl transferase (OT), the enzyme that catalyzes the cotranslational glycosylation of asparagine [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637; Kellenberger, C., Hendrickson, T. L., and Imperiali, B. (1997) Biochemistry 36, 12554-12559]. However, no specific OT inhibitors that function in the cellular environment have yet been reported. The peptide cyclo(hex-Amb-Cys)-Thr-Val-Thr-Nph-NH2 was previously shown to exhibit nanomolar inhibition (Ki = 37 nM) through slow tight binding kinetics [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637]. Included herein is the redesign of this prototype inhibitor for achieving both passive and active translocation into model membrane systems representing the endoplasmic reticulum (ER). The strategy for passive transport involved the incorporation of a membrane permeable import function previously shown to carry various peptides across the outer as well as the interior cellular membranes [Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y.-Z. (1998) Nat. Biotechnol. 16, 370-375]. Assessment of function in intact ER membranes revealed that the inhibitor targeted toward passive diffusion demonstrated concentration-dependent inhibition of two different glycosylation substrates. Thus, this modified inhibitor achieved potent inhibition of glycosylation after being successfully transported through the ER membrane. In the active translocation approach, the lead OT inhibitor and a corresponding substrate were redesigned to include features recognized by the transporter associated with antigen processing (TAP). This protein translocates peptides into the lumen of the ER [Heemels, M.-T., Schumacher, T. N. M., Wonigeit, K., and Ploegh, H. L. (1993) Science 262, 2059-2063]. However, although acceptance of the cyclized substrate by the TAP receptor was demonstrated via efficient transport and glycosylation, the modified inhibitor was not translocated by TAP machinery, and therefore, active translocation was achieved for the modified substrate only. Both of these ER transport methods afforded redesigned OT inhibitors that retained their inhibitor properties in vitro, regardless of the extensions to the carboxy-terminus of the root inhibitor. The above family of redesigned inhibitors provides a template for generating a transcellular pathway and represents the first step toward OT inhibition in intact cells.     

Role of zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase

In previous studies [Delker, S. L., et al. (2010), J. Am. Chem. Soc. 132, 5437-5442], we determined the crystal structures of neuronal nitric oxide synthase (nNOS) in complex with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S,S)-cis inhibitors, an unexpected "flipped" orientation was observed for the (R,R)-cis enantiomers. In the flipped mode, the aminopyridine extends out of the active site where it interacts with one heme propionate. This prompted us to design and synthesize symmetric "double-headed" inhibitors with an aminopyridine at each end of a bridging ring structure [Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J., Marta?sek, P., Roman, L. J., Poulos, T. L., and Silverman, R. B. Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase. J. Med. Chem. (submitted for publication)]. One aminopyridine should interact with the active site Glu and the other with the heme propionate. Crystal structures of these double-headed aminopyridine inhibitors in complexes with nNOS show unexpected and significant protein and heme conformational changes induced by inhibitor binding that result in removal of the tetrahydrobiopterin (H(4)B) cofactor and creation of a new Zn(2+) site. These changes are due to binding of a second inhibitor molecule that results in the displacement of H(4)B and the placement of the inhibitor pyridine group in position to serve as a Zn(2+) ligand together with Asp, His, and a chloride ion. Binding of the second inhibitor molecule and generation of the Zn(2+) site do not occur in eNOS. Structural requirements for creation of the new Zn(2+) site in nNOS were analyzed in detail. These observations open the way for the potential design of novel inhibitors selective for nNOS.     

Arresting initiation of hepatitis C virus RNA synthesis using heterocyclic derivatives

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. Recently, two benzo-1,2,4-thiadiazine compounds were shown to be potent, highly specific inhibitors of the genotype 1b HCV RdRp containing a carboxyl-terminal 21 residue truncation (delta21 HCV RdRp) (Dhanak, D., Duffy, K., Johnston, V. K., Lin-Goerke, J., Darcy, M., Shaw, A. N. G. B., Silverman, C., Gates, A. T., Earnshaw, D. L., Casper, D. J., Kaura, A., Baker, A., Greenwood, C., Gutshall, L. L., Maley, D., DelVecchio, A., Macarron, R., Hofmann, G. A., Alnoah, Z., Cheng, H.-Y., Chan, G., Khandekar, S., Keenan, R. M., and Sarisky, R. T. (2002) J. Biol. Chem. 277, 38322-38327). Compound 4 (C(21)H(21)N(3)O(4)S) reduces viral replication by virtue of its direct interaction with the viral polymerase rather than by nonspecific titration of nucleic acid template. In this study, we present several lines of evidence to demonstrate that this inhibitor interferes with the initiation step of RNA synthesis rather than acting as an elongation inhibitor. Inhibition of initial phosphodiester bond formation occurred regardless of whether replication was initiated by primer-dependent or de novo mechanisms. Filter binding studies using increasing concentrations of compound 4 did not interfere with the ability of delta21 HCV RdRp to interact with nucleic acid. Furthermore, varying the order of reagent addition in the primer extension assay showed no distinct differences in inhibition profile. Finally, surface plasmon resonance analyses provided evidence that a ternary complex is capable of forming between the RNA template, RdRp, and compound 4. Together, these data suggest that this heterocyclic agent interacts with the apoenzyme, as well as with the RNA-bound form of delta21 HCV RdRp, and therefore does not directly interfere with the RdRp-RNA interaction to mediate inhibition.     

Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the beta-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., & Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 +/- 0.02 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adaptation of acyl-enzyme kinetic theory and an experimental method for evaluating the kinetics of fast-acting, irreversible protease inhibitors

The theory of acyl-enzyme kinetics (Bender, M.L., Kézdy, F.J. and Wedler, F.C. (1967) J. Chem. Educ. 44, 84-88) has been adapted for use in evaluating the kinetics of inhibition of serine proteases by both natural and synthetic irreversible inhibitors. The new theory is based upon formal analysis of the case of an irreversible, active-site-directed inhibitor competing with an irreversible, active-site-directed substrate for the active site of a serine protease. From this theory, an experimentally simple and accurate method is described to obtain a second-order rate constant that is characteristic of the efficiency with which an irreversible inhibitor reacts. The experimental method is particularly useful for characterizing fast-acting, irreversible inhibitors. The theory and method which are applicable to a wide variety of enzymes are verified by analysis of the inhibition of bovine trypsin by three model inhibitors, p-nitrophenyl p'-guanidinobenzoate, soybean trypsin inhibitor and alpha-1-proteinase inhibitor as well as by human antithrombin III in the presence of heparin and by bovine pancreatic trypsin inhibitor.     

Interaction of anions with the active site of carboxypeptidase A

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic dissection of the catalytic mechanism of taurine:alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli

Recent studies on taurine:alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli have provided evidence for a three-step, minimal kinetic mechanism involving the quaternary TauD.Fe(II).alpha-ketoglutarate.taurine complex, the taurine-hydroxylating Fe(IV)-oxo intermediate (J) that forms upon reaction of the quaternary complex with O(2), and a poorly defined, Fe(II)-containing intermediate state that converts in the rate-limiting step back to the quaternary complex [Price, J. C., Barr, E. W., Tirupati, B., Bollinger, J. M., Jr., and Krebs, C. (2003) Biochemistry 42, 7497-7508]. The mapping of this kinetic mechanism onto the consensus chemical mechanism for the Fe(II)- and alpha-ketoglutarate-dependent engendered several predictions and additional questions that have been experimentally addressed in the present study. The results demonstrate (1) that postulated intermediates between the quaternary complex and J accumulate very little or not at all; (2) that decarboxylation of alpha-ketoglutarate occurs prior to or concomitantly with formation of J; (3) that the second intermediate state comprises one or more product complex with Mossbauer features that are partially resolved from those of the binary TauD.Fe(II), ternary TauD.Fe(II).alpha-ketoglutarate, and quaternary TauD.Fe(II).alpha-ketoglutarate.taurine complexes; and (4) that the rate-determining step in the catalytic cycle is release of product(s) prior to the rapid, ordered binding of alpha-ketoglutarate and then taurine to regenerate the O(2)-reactive quaternary complex. The results thus integrate the previously proposed kinetic and chemical mechanisms and indicate which of the postulated intermediates in the latter will be detectable only upon perturbation of the kinetics by changes in reaction conditions (e.g., temperature), protein mutagenesis, the use of substrate analogues, or some combination of these.     

Type II Transmembrane Serine Proteases

Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857–860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field.