Alternate Substrate Inhibitors Research Articles

Mechanism of germination inhibition of Clostridioides difficile spores by an aniline substituted cholate derivative (CaPA).

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea and has been declared an urgent threat by the CDC. C. difficile forms dormant and resistant spores that serve as infectious vehicles for CDI. To cause disease, C. difficile spores recognize taurocholate and glycine to trigger the germination process. In contrast to other sporulating bacteria, C. difficile spores are postulated to use a protease complex, CspABC, to recognize its germinants. Since spore germination is required for infection, we have developed anti-germination approaches for CDI prophylaxis. Previously, the bile salt analog CaPA (an aniline-substituted cholic acid) was shown to block spore germination and protect rodents from CDI caused by multiple C. difficile strains and isolates. In this study, we found that CaPA is an alternative substrate inhibitor of C. difficile spore germination. By competing with taurocholate for binding, CaPA delays C. difficile spore germination and reduces spore viability, thus diminishing the number of outgrowing vegetative bacteria. We hypothesize that the reduction of toxin-producing bacterial burden explains CaPA's protective activity against murine CDI. Previous data combined with our results suggests that CaPA binds tightly to C. difficile spores in a CspC-dependent manner and irreversibly traps spores in an alternative, time-delayed, and low yield germination pathway. Our results are also consistent with kinetic data suggesting the existence of at least two distinct bile salt binding sites in C. difficile spores.

Identification of Infigratinib as a Potent Reversible Inhibitor and Mechanism-Based Inactivator of CYP2J2: Nascent Evidence for a Potential In Vivo Metabolic Drug-Drug Interaction with Rivaroxaban.

Infigratinib (INF) is a fibroblast growth factor receptor inhibitor that was recently United States Food and Drug Administration-approved for the treatment of advanced or metastatic cholangiocarcinoma. We previously established that INF inhibited and inactivated cytochrome P450 3A4 (CYP3A4). Here, in a follow up to our previous study, we identified for the first time that INF also elicited potent competitive inhibition and mechanism-based inactivation of CYP2J2 with kinetic parameters K i, K I, k inact, and a partition ratio of 1.94 µM, 0.10 µM, 0.026 minute-1, and ∼3, respectively, when rivaroxaban was harnessed as the probe substrate. Inactivation was revealed to exhibit cofactor-dependency and was attenuated by an alternative substrate (astemizole) and direct inhibitor (nilotinib) of CYP2J2. Additionally, the nature of inactivation was unlikely to be pseudo-irreversible and instead arose from covalent modification due to the lack of substantial enzyme activity recovery after dialysis and chemical oxidation, as well as the lack of a resolvable Soret band in spectral scans. Glutathione trapping confirmed that the identity of the putative reactive intermediate implicated in the covalent inactivation of both CYP2J2 and CYP3A4 was identical and likely attributable to an electrophilic p-benzoquinonediimine intermediate of INF. Finally, mechanistic static modeling revealed that by integrating the previously arcane inhibition and inactivation kinetic parameters of CYP2J2-mediated rivaroxaban hydroxylation by INF illuminated in this work, together with those previously documented for CYP3A4, a 49% increase in the systemic exposure of rivaroxaban was projected. Our modeling results predicted a potential risk of metabolic drug-drug interactions between the clinically relevant combination of rivaroxaban and INF in the setting of cancer. SIGNIFICANCE STATEMENT: This study reported that INF elicits potent reversible inhibition and mechanism-based inactivation of CYP2J2. Furthermore, static modelling predicted that its coadministration with the direct oral anticoagulant rivaroxaban may potentially culminate in a metabolic drug-drug interaction (DDI) leading to an increased risk of major bleeding. As rivaroxaban is steadily gaining prominence as the anticoagulant of choice in the treatment of cancer-associated venous thromboembolism, the DDI projections reported here are clinically relevant and warrant further investigation via physiologically based pharmacokinetic modelling and simulation.

Direct and Sequential Bioactivation of Pemigatinib to a Reactive Iminium Ion Intermediate Culminates in Mechanism‐Based Inactivation of Cytochrome P450 3A

Mechanism‐based inactivation (MBI) is a unique phenomenon in drug metabolism that arises when an enzyme metabolically activates a drug to a chemically reactive intermediate and primes it for irreversible binding, leading to an irrevocable loss of catalytic activity that is only restored upon de novo synthesis of the implicated enzyme. Our laboratory recently established the MBI of cytochrome P450 3A (CYP3A) by the fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and infigratinib. Serendipitously, our preliminary data has also revealed that pemigatinib (PEM) – another clinically approved FGFR1‐3 inhibitor – similarly elicited time‐dependent inhibition of CYP3A. This was rather unexpected as it was previously purported that PEM did not pose any metabolism‐dependent liabilities due to the absence of glutathione‐related conjugates in metabolic profiling experiments conducted in human liver microsomes. Consequently, we posited that PEM is bioactivated by CYP3A to a hard electrophile, thereby allowing it to elude glutathione trapping and detection in the aforementioned reactive metabolite screen. Here, we confirmed that PEM inhibited both CYP3A isoforms in a time‐, concentration‐, and cofactor‐dependent manner consistent with MBI – with KI, kinact, and partition ratio of 8.69 and 11.95 μM, 0.108 and 0.042 min−1, and ~44 and ~47 for CYP3A4 and CYP3A5 respectively, when rivaroxaban was employed as the probe substrate. While the rate of inactivation was diminished by coincubation with an alternative substrate (testosterone) or direct inhibitor of CYP3A (ketoconazole), the inclusion of nucleophilic trapping agents afforded no such protection. Furthermore, the lack of catalytic activity recovery following dialysis and oxidation with potassium ferricyanide coupled with the absence of a spectrally resolvable peak in the Soret region collectively implied that the underlying mechanism of inactivation was not elicited via the formation of pseudoirreversible metabolite‐intermediate complexes. Utilizing cyanide trapping and high‐resolution mass spectrometry, we illuminated the direct and sequential oxidative bioactivation of PEM and its major O‐desmethylated metabolite at its distal morpholine moiety to a reactive iminium ion intermediate that could covalently modify the CYP3A apoprotein and/or prosthetic heme and culminate in its MBI. Finally, structural determinants gleaned from our in silicocovalent docking simulations suggested that the discrepancies in MBI potencies between both CYP3A isoforms might be alluded to the propensity for PEM to interact more favourably with an accessible active‐site serine residue in position 312 of CYP3A4 as compared to CYP3A5. Further physiologically based pharmacokinetic modelling studies are currently underway to better discern the clinical implications of our findings.

Direct and Sequential Bioactivation of Pemigatinib to Reactive Iminium Ion Intermediates Culminates in Mechanism-Based Inactivation of Cytochrome P450 3A.

We recently established the mechanism-based inactivation (MBI) of cytochrome P450 3A (CYP3A) by the fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and infigratinib. Serendipitously, our preliminary data have also revealed that pemigatinib (PEM), another clinically approved FGFR1-3 inhibitor, similarly elicited time-dependent inhibition of CYP3A. This was rather unexpected, as it was previously purported that PEM did not pose any metabolism-dependent liabilities due to the absence of glutathione-related conjugates in metabolic profiling experiments conducted in human liver microsomes. Here, we confirmed that PEM inhibited both CYP3A isoforms in a time-, concentration-, and cofactor-dependent manner consistent with MBI, with inactivator concentration at half-maximum rate constant, maximum inactivation rate constant, and partition ratio of 8.69 and 11.95 μM, 0.108 and 0.042 min-1, and approximately 44 and approximately 47 for CYP3A4 and CYP3A5, respectively. Although the rate of inactivation was diminished by coincubation with an alternative substrate or direct inhibitor of CYP3A, the inclusion of nucleophilic trapping agents afforded no such protection. Furthermore, the lack of catalytic activity recovery following dialysis and oxidation with potassium ferricyanide coupled with the absence of a spectrally resolvable peak in the Soret region collectively implied that the underlying mechanism of inactivation was not elicited via the formation of pseudo-irreversible metabolite-intermediate complexes. Finally, utilizing cyanide trapping and high-resolution mass spectrometry, we illuminated the direct and sequential oxidative bioactivation of PEM and its major O-desmethylated metabolite at its distal morpholine moiety to reactive iminium ion hard electrophilic species that could covalently inactivate CYP3A via MBI. SIGNIFICANCE STATEMENT: This study reports for the first time the covalent MBI of CYP3A by PEM and deciphered its bioactivation pathway involving the metabolic activation of PEM and its major O-desmethylated metabolite to reactive iminium ion intermediates. Following which, a unique covalent docking methodology was harnessed to unravel the structural and molecular determinants underpinning its inactivation. Findings from this study lay the foundation for future investigation of clinically relevant drug-drug interactions between PEM and concomitant substrates of CYP3A.

Mechanism-Based Inactivation of Cytochrome P450 3A4 and 3A5 by the Fibroblast Growth Factor Receptor Inhibitor Erdafitinib.

Erdafitinib (ERD) is a first-in-class pan inhibitor of fibroblast growth factor receptor 1-4 that has garnered global regulatory approval for the treatment of advanced or metastatic urothelial carcinoma. Although it has been previously reported that ERD elicits time-dependent inhibition (TDI) of cytochrome P450 (P450) 3A4 (CYP3A4), the exact biochemical nature underpinning this observation remains obfuscated. Moreover, it is also uninterrogated if CYP3A5-its highly homologous counterpart-could be susceptible to such interactions. Mechanism-based inactivation (MBI) of P450 is a unique subset of TDI that hinges on prior bioactivation of the drug to a reactive intermediate and possesses profound clinical and toxicological implications due to its irreversible nature. Here, we investigated and confirmed that ERD inactivated both CYP3A isoforms in a time-, concentration-, and NADPH-dependent manner with KI, kinact, and partition ratio of 4.01 and 10.04 μM, 0.120 and 0.045 min-1, and 32 and 55 for both CYP3A4 and CYP3A5, respectively, when rivaroxaban was employed as the probe substrate. Co-incubation with an alternative substrate or direct inhibitor of CYP3A attenuated the rate of inactivation, whereas the addition of glutathione or catalase did not induce such protection. The lack of enzyme activity recovery following dialysis for 4 h and oxidation with potassium ferricyanide combined with the lack of a Soret peak in spectral scans collectively substantiated that ERD is an irreversible covalent MBI of CYP3A. Finally, glutathione trapping and high-resolution mass spectrometry experiments illuminated a plausible bioactivation mechanism of ERD by CYP3A arising from metabolic epoxidation of its quinoxaline ring.

Design, synthesis and antiviral evaluation of 2'-C-methyl branched guanosine pronucleotides: the discovery of IDX184, a potent liver-targeted HCV polymerase inhibitor.

Ribonucleoside analogs possessing a β-methyl substituent at the 2'-position of the d-ribose moiety have been previously discovered to be potent and selective inhibitors of hepatitis C virus (HCV) replication, their triphosphates acting as alternative substrate inhibitors of the HCV RdRp NS5B. Results/methodology: In this article, the authors detail the synthesis, anti-HCV evaluation in cell-based replicon assays and structure-activity relationships of several phosphoramidate diester derivatives of 2'-C-methylguanosine (2'-MeG). The most promising compound, namely the O-[S-(hydroxyl)pivaloyl-2-thioethyl]{abbreviated as O-[(HO)tBuSATE)]} N-benzylamine phosphoramidate diester derivative (IDX184), was selected for further in vivo studies, and was the first clinical pronucleotide evaluated for the treatment of chronic hepatitis C up to Phase II trials.

(399) - Emerging Combination Activity and Resistance Profile for Brincidofovir (CMX001) in CMV Prevention and Treatment

Brincidofovir (CMX001, BCV) is a broad-spectrum oral antiviral in Phase 3 development for prevention of CMV in hematopoietic cell transplantation. BCV is converted intracellularly to cidofovir diphosphate which acts as an alternative substrate inhibitor for the CMV viral DNA polymerase (UL54, pol). The combination activity and resistance profile of BCV was evaluated in vitro and in two completed clinical trials; Study 201enrolled CMV antiviral naïve subjects and Study 350 enrolled antiviral therapy experienced patients.

Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA

PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2'-F-2'-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2'-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type.

Nucleoside Analog Inhibitors of Hepatitis C Viral Replication: Recent Advances, Challenges and Trends

Chronic hepatitis C virus (HCV) infection is a global health problem, with over 170 million people infected worldwide. The current therapy, pegylated interferon (PEG-IFN) plus ribavirin (RBV), provides only approximately a 40% sustained virological response (undetectable HCV RNA for greater than 24 weeks after cessation of therapy), in genotype 1-infected individuals. In addition to the limited sustained virological response, PEG-IFN/RBV treatment is associated with serious adverse effects. Nucleosides have long been the cornerstone of antiviral therapy because of their proven efficacy and high barrier to resistance. Through the use of surrogate viruses or the HCV subgenomic replicon, several classes of nucleoside analogs or their monophosphate prodrugs have been identified that inhibit HCV RNA replication. Nucleoside analogs that possess the 2´-C-methyl modification vary in their ability to be phosphorylated and to act as alternative substrate inhibitors of the HCV RNA polymerase. Herein, we discuss various classes of nucleoside inhibitors, with a focus on available structure-activity relationships, their mode of action and resistance profile.

QSAR for Inhibition of Pseudomonas Species Lipase by 1‐Acyloxy‐3‐N‐n‐octylcarbamyl‐benzenes

Abstract1‐Acyloxy‐3‐N‐n‐octylcarbamyl‐benzenes (1–9) are synthesized to characterize the Quantitative Structure–Activity Relationship (QSAR) for the Third Acyl Group Binding Site (TACS) of Pseudomonas species lipase. Inhibitors 1–9 are characterized as pseudo or alternate substrate inhibitors of the enzyme. The inhibition constant (Ki) and carbamylation constant (k2) for the enzyme inhibitions by inhibitors 1–9 are determined. The carbamate carbons of the n‐octylcarbamyl moieties of inhibitors 1–9 are nucleophilically attacked by the active site serine of the enzyme and the n‐octylcarbamyl groups of inhibitors 1–9 are bound to the Acyl Group Binding Site (ACS) of the enzyme. Both pKi and log k2 values are linearly corrected with the Hansch hydrophobicity π values of the substituents of the acyl moieties of inhibitors 1–7. The slopes for these corrections are 0.13 and 0.02, respectively. This result suggests that the enzyme inhibitions by inhibitors 1–7 have a common mechanism. Thus, all acyl moieties of inhibitors 1–7 should bind to the TACS of the enzyme since the acyl and carbamyl moieties of inhibitors 1–7 are meta to each other. This result also indicates that the major interaction between the acyl moiety of inhibitors 1–7 and the TACS of the enzyme is primarily the hydrophobic interaction. The more hydrophobic characters of inhibitors 1–7 are, the more tightly these inhibitors bind to the enzyme. In contrast, 1‐triphenylacetoxy‐3‐N‐n‐octylcarbamyl‐benzene (8) and 1‐trimethylacetoxy‐3‐N‐n‐octylcarbamyl‐benzene (9) do not bind to the TACS of the enzyme due to the fact that the inhibitions by both inhibitors are not linearly correlated with π. It is possible that these two inhibitors are too bulky to fit into the TACS of the enzyme.

590] INHIBITION OF HCV REPLICATION BY PSI-6130: MECHANISM OF BIOCHEMICAL ACTIVATION AND INHIBITION

Background: Nearly 2% of the U S . population and an estimated 170 million people worldwide are HCV carriers. The current standard of care, a combination of pegylated interferon and ribavirin, has limited efficacy. We have identified a nucleoside analog, (3-D-2’-deoxy-2’-fluoro2’-C-methylcytidine (PSI-61 30), which is a specific, potent and noncytotoxic inhibitor of HCV in the subgenomic replicon assay. To inhibit the HCV NS5B polymerase, PSI-6 130 must be phosphorylated to the 5’triphosphate. The enzymes involved in the phosphorylation of PSI-61 30 and inhibition of HCV NS5B were investigated. Methods: Anti-HCV activity was measured using subgenomic-HCV replicon cells and a real-time PCR assay. Spectrophotometric assays and recombinant deoxycytidine kinase, uridine-cytidine kinase, UMP-CMP kinase, and nucleoside diphosphate kinase (NDPK) were used to investigate the phosphorylation pathway of PSI-6130 to the corresponding 5’-triphosphate. The NS5B RNA polymerase reaction was performed in the presence of PSI-6 130-TP or other 2’-C-Me-nucleoside triphosphates using 32P-labeled UTP and the amount of radioactivity in the product was measured. Results: An EC90 (concentration producing I loglo reduction in viral RNA) value of 4.6f2.0pM was determined for PSI-6130 and was essentially inactive against other viruses including BVDV Little or no cytotoxicity or mitochondrial toxicity was observed with cells treated with PSI-6 130. Purified dCK, UMP-CMP kinase, and NDPK phosphorylated PSI-61 30 to the 5’-mono-, di-, and triphosphate derivatives respectively. PSI-6130-TP was an alternative substrate inhibitor of purified NSSB. Incorporation of PSI-6130-MP into RNA catalyzed by purified NS5B resulted in chain-termination. The steady-state inhibition constant for PSI6 130-TP was Ki = 4.3 pM. Against the S282T NS5B enzyme PSI-6 130-TP showed only a 3-fold resistance compared to a 12-fold resistance for 2‘C-Me-cytidine-TP and a 150-fold resistance for 2’-C-Me-adenosine-TP. Conclusion: PSI-6 130 demonstrated potent and specific anti-HCV activity in cell-based assays with little or no cytotoxicity and mitochondrial toxicity. PSI-6 130 was efficiently phosphorylated to its active 5’-triphosphate form. Deoxycytidine kinase, UMP-CMP kinase, and NDPK were identified as enzymes involve in the phosphorylation pathway of PSI-6130. PSI6130-TP inhibited HCV NSSB RNA polymerase and acted as a chainterminator upon incorporation into the elongating RNA strand by HCV NS5B.

Synthesis of Tricyclic 1,3-Oxazin-4-ones and Kinetic Analysis of Cholesterol Esterase and Acetylcholinesterase Inhibition

A series of thieno[1,3]oxazin-4-ones and thieno[1,3]thiazin-4-ones were synthesized and investigated as inhibitors of the alpha/beta hydrolases cholesterol esterase (CEase) and acetylcholinesterase (AChE). The introduction of a cycloaliphatic five- or six-membered ring fused at the thiophene was favorable for CEase inhibition. Such compounds were analyzed as true alternate substrate inhibitors. 6,7-Dihydro-2-(dimethylamino)-4H,5H-cyclopenta[4,5]thieno[2,3-d][1,3]oxazin-4-one (33) exhibited a K(i) value of 630 nM and excelled in its low susceptibility to CEase-catalyzed degradation. Compound 33 and its analogues did not inhibit AChE. The introduction of a tetrahydropyrido ring with bulky hydrophobic substituents at the basic nitrogen provided inhibitors of AChE which were completely inactive toward CEase. 7-Benzyl-5,6,7,8-tetrahydro-2-(N-3,4-dimethoxybenzyl-N-methylamino)-4H-pyrido[4',3':4,5]thieno[2,3-d][1,3]oxazin-4-one (21) had the IC(50) value of 330 nM for AChE inhibition. A residual enzymatic activity at an infinite inhibitor concentration and thus a catalytically active ternary enzyme-substrate-inhibitor complex was concluded. To specify kinetic parameters of inhibition, a new method was derived to characterize selected thieno[1,3]oxazin-4-ones as hyperbolic mixed-type inhibitors of AChE.

Recent developments in the design of mechanism-based and alternate substrate inhibitors of serine proteases.

A wide range of human diseases are associated with the aberrant activity of mammalian, viral, bacterial or parasitic proteases. These include members of all four classes of proteases, namely, serine, cysteine, aspartic and metallo- proteases. The involvement of proteases in disease states has provided the impetus behind studies related to the design of potent and selective inhibitors and their use as either therapeutic agents and/or pharmacological probes to gain a better understanding of the pathophysiology of a particular disease. This review focuses on recent developments related to the design of mechanism-based and alternate substrate inhibitors of serine proteases of mammalian and non-mammalian origin. Numerous examples are cited that illustrate the fundamental principles and subtleties associated with the design of covalent and non-covalent inhibitors of these enzymes. This is an exciting and promising area of investigation that will undoubtedly continue unabated in the future.

Mutation of active site residues of the puromycin-sensitive aminopeptidase: conversion of the enzyme into a catalytically inactive binding protein

The active site glutamate, Glu 309, of the puromycin-sensitive aminopeptidase was mutated to glutamine, alanine, and valine. These mutants were characterized with amino acid β-naphthylamides as substrates and dynorphin A(1-9) as an alternate substrate inhibitor. Conversion of glutamate 309 to glutamine resulted in a 5000- to 15,000-fold reduction in catalytic activity. Conversion of this residue to alanine caused a 25,000- to 100,000-fold decrease in activity, while the glutamate to valine mutation was the most dramatic, reducing catalytic activity 300,000- to 500,000-fold. In contrast to the dramatic effect on catalysis, all three mutations produced relatively small (1.5- to 4-fold) effects on substrate binding affinity. Mutation of a conserved tyrosine, Y394, to phenylalanine resulted in a 1000-fold decrease in k cat, with little effect on binding. Direct binding of a physiological peptide, dynorphin A(1-9), to the E309V mutant was demonstrated by gel filtration chromatography. Taken together, these data provide a quantitative assessment of the effect of mutating the catalytic glutamate, show that mutation of this residue converts the enzyme into an inactive binding protein, and constitute evidence that this residue acts a general acid/base catalyst. The effect of mutating tyrosine 394 is consistent with involvement of this residue in transition state stabilization.

Alternate Substrate Inhibition of Cholesterol Esterase by Thieno[2,3-d][1,3]oxazin-4-ones

In a kinetic study, the interaction of bovine pancreatic cholesterol esterase (CEase) with fused 1,3-oxazin-4-ones and 1,3-thiazin-4-ones was investigated, and the compounds were characterized as alternate substrate inhibitors. Inhibition assays were performed in the presence of sodium taurocholate with p-nitrophenyl butyrate as chromogenic substrate. Strong active site-directed inhibition was detected for 2-diethylaminothieno[2,3-d][1,3]oxazin-4-ones with a cycloaliphatic chain at positions 5,6. The most potent inhibitors, compounds 3 and 4, exhibited K(i) values of 0.58 and 1.86 microm, respectively. An exchange of the ring oxygen by sulfur and the removal of the cycloaliphatic moiety as well as the replacement of the thiophene ring by benzene led to a loss of inhibitory potency. CEase has the capability to catalyze the hydrolysis of representatives of fused 1,3-oxazin-4-ones as well as the highly stable 1,3-thiazin-4-ones by using an acylation-deacylation mechanism. Hydrolyses were performed in the presence of a high enzyme concentration, and products were identified spectrophotometrically and by means of high performance liquid chromatography. The kinetic parameters V(max)I and V(max)I/K(m)(I) for the CEase-catalyzed turnover were determined. The compounds whose enzyme-catalyzed hydrolysis followed first-order kinetics (K(m)(I) > 25 microm) failed to inhibit CEase. On the other hand, inhibitors 3 (initial concentration of 25 microm) and 4 (20 microm) were hydrolyzed by CEase under steady-state conditions in the first phase of the reaction. Rate-limiting deacylation was demonstrated in nucleophilic competition experiments with ethanol as acyl acceptor wherein the conversion of compound 3 was accelerated up to an ethanol concentration of 1.5 m. The characterization of these compounds (i.e. 3 and 4) as alternate substrate inhibitors is not only based on the verification of the CEase-catalyzed hydrolysis. It also rests upon the concurrence of corresponding K(i) values obtained in the inhibition assay compared with separately determined K(m)(I) values of their enzyme-catalyzed consumption, as could be predicted from the kinetic model used in this study.

Mechanism of action of 1-beta-D-2,6-diaminopurine dioxolane, a prodrug of the human immunodeficiency virus type 1 inhibitor 1-beta-D-dioxolane guanosine.

(-)-beta-D-2,6-Diaminopurine dioxolane (DAPD), is a nucleoside reverse transcriptase (RT) inhibitor with activity against human immunodeficiency virus type 1 (HIV-1). DAPD, which was designed as a water-soluble prodrug, is deaminated by adenosine deaminase to give (-)-beta-D-dioxolane guanine (DXG). By using calf adenosine deaminase a K(m) value of 15 +/- 0.7 microM was determined for DAPD, which was similar to the K(m) value for adenosine. However, the k(cat) for DAPD was 540-fold slower than the k(cat) for adenosine. In CEM cells and peripheral blood mononuclear cells exposed to DAPD or DXG, only the 5'-triphosphate of DXG (DXG-TP) was detected. DXG-TP is a potent alternative substrate inhibitor of HIV-1 RT. Rapid transient kinetic studies show the efficiency of incorporation for DXG-TP to be lower than that measured for the natural substrate, 2'-deoxyguanosine 5'-triphosphate. DXG-TP is a weak inhibitor of human DNA polymerases alpha and beta. Against the large subunit of human DNA polymerase gamma a K(i) value of 4.3 +/- 0.4 microM was determined for DXG-TP. DXG showed little or no cytotoxicity and no mitochondrial toxicity at the concentrations tested.

Recent advances in antiviral research : identification of inhibitors of the herpesvirus proteases

Major advances have been reported in the last two years regarding the molecular biology and structural properties of the herpesvirus proteases. X-ray diffraction studies have enabled several groups to solve the structure of the human cytomegalovirus protease. Fluorescence-based substrate assays have also been recently reported. These substrates exhibit sufficient kinetic and sensitivity properties to enable high-throughput screening efforts dedicated toward the discovery of protease inhibitors. Three classes of inhibitors have been reported recently: nonpeptidic aryl trifluoromethylketones; alternate substrate inhibitors (benzoxazinones/azalactones); and thiol-modifying inhibitors. The thiol-modifying class offers a unique opportunity to discover inhibitors specific to the human cytomegalovirus protease, as this protease requires reduced cysteine residues for its enzymatic activity.

Biosynthesis of the Macrolide Oleandomycin by Streptomyces antibioticus: PURIFICATION AND KINETIC CHARACTERIZATION OF AN OLEANDOMYCIN GLUCOSYLTRANSFERASE

The oleandomycin (OM) producer, Streptomyces antibioticus, possesses a mechanism involving two enzymes for the intracellular inactivation and extracellular reactivation of the antibiotic. Inactivation takes place by transfer of a glucose molecule from a donor (UDP-glucose) to OM, a process catalyzed by an intracellular glucosyltransferase. Glucosyltransferase activity is detectable in cell-free extracts concurrent with biosynthesis of OM. The enzyme has been purified 1,097-fold as a monomer, with a molecular mass of 57.1 kDa by a four-step procedure using three chromatographic columns. The reaction operates via a compulsory-order mechanism. This has been shown by steady-state kinetic studies using either OM or an alternative substrate (rosaramycin) and dead-end inhibitors, and isotopic exchange reactions at equilibrium. OM binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated oleandomycin (GS-OM).

Degradation of Dynorphin‐Related Peptides by the Puromycin‐Sensitive Aminopeptidase and Aminopeptidase M

The degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. Ki determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin-related peptides, i.e., Ki for dynorphin A-17 = 23-30 nM with the Ki increasing to 25-50 microM for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, kcat remains relatively constant; however, as the peptide size increases beyond a decapeptide, kcat decreases significantly. With the puromycin-sensitive aminopeptidase, similar results were obtained except that kcat was greatest for the pentapeptide. Thus, if one considers kcat/Km as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin-sensitive aminopeptidase in the degradation of extended dynorphin-related peptides.

(3R, 4S)-3-Benzyl-4-bromomethyloxetan-2-one, a fast acting alternate substrate inhibitor of α-chymotrypsin

(3 R,4 S)-3-Benzyl-4-(bromomethyl)oxetan-2-one is a novel type of fast acting alternate substrate inhibitor for α-chymotrypsin. The hydroxyl group that is generated during the turnover process appears to be responsible for the high stability of the acyl enzyme intermediate.