Peptidomimetic Inhibitors of the Human Cytomegalovirus Protease

Ogilvie, William W.; Bailey, Murray; Poupart, Marc‐André; Abraham, Robin; Bhavsar, Amit P.; Bonneau, Pierre; Bordeleau, Josée; Bousquet, Yves; Chabot, Catherine; Duceppe, Jean‐Simon; Fazal, Gulrez; Goulet, Sylvie; Grand‐Maître, Chantal; Guse, Ingrid; Halmos, Ted; Lavallée, Pierre; Leach, Michael; Malenfant, Éric; O'Meara, Jeff A.; Plante, Raymond; Plouffe, Céline; Poirier, Martin; Soucy, François; Yoakim, Christiane; Déziel, Robert

doi:10.1021/jm970104t

Cited by 94 publications

(101 citation statements)

References 42 publications

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“…Accordingly, norleucine was replaced by glutamine to improve solubility. Additionally, previous results suggest a preference for tert-butylglycine at the P3 position, so this information was also incorporated into the modified substrate (35). Kinetic analysis of the tetrameric ACC substrate revealed Michaelis-Menten kinetics with a K m of 81 Ϯ 11 M and a k cat of 0.022 Ϯ 0.001 s Ϫ1 , a dramatic improvement over the intramolecularly quenched substrate previously used in enzymatic assays (24).…”

Section: Resultsmentioning

confidence: 92%

Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor

Marnett

Nomura²,

Shimba³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Structurally diverse organophosphonate inhibitors targeting the active site of the enzyme were used to investigate the relationship of the active site and the dimer interface of wild-type protease in solution. Positional scanning synthetic combinatorial libraries revealed Kaposi's sarcoma-associated herpesvirus protease to be highly specific, even at sites distal to the peptide bond undergoing hydrolysis. Specificity results were used to synthesize a hexapeptide diphenylphosphonate inhibitor of Kaposi's sarcoma-associated herpesvirus protease. The transition state analog inhibitors covalently phosphonylate the active site serine, freezing the enzyme structure during catalysis. An NMR-based assay was developed to monitor the native monomer-dimer equilibrium in solution and was used to demonstrate the effect of protease inhibition on the quaternary structure of the enzyme. NMR, circular dichroism, and size exclusion chromatography analysis showed that active site inhibition strongly regulates the binding affinity of the monomer-dimer equilibrium at the spatially separate dimer interface of the protease, shifting the equilibrium to the dimeric form of the enzyme. Furthermore, inhibitor studies revealed that the catalytic cycles of the spatially separate active sites are independent. These results (i) provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, (ii) establish a molecular mechanism of protease activation and stabilization during catalysis, and (iii) highlight potential implications of substoichiometric inhibition of the viral protease in developing herpesviral therapeutics.

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Section: Resultsmentioning

confidence: 92%

Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor

Marnett

Nomura²,

Shimba³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Selectivity Serine Protease Assays-Bovine pancreatic ␣-chymotrypsin and human leukocyte elastase (HLE) were obtained from Roche Applied Science and Calbiochem and assayed as described previously (29).…”

Section: Methodsmentioning

confidence: 99%

An NS3 Serine Protease Inhibitor Abrogates Replication of Subgenomic Hepatitis C Virus RNA

Pause¹,

Kukolj²,

Bailey

et al. 2003

Journal of Biological Chemistry

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The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-␣ (IFN-␣), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-␣ showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-␣ also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection. HCV1 as a member of the Flaviviridae family is the major etiological agent of non-A, non-B viral hepatitis and an important cause of chronic liver disease leading to cirrhosis and hepatocellular carcinoma in humans (1, 2). An estimated 170 million people worldwide are infected with HCV, and end stage liver disease associated with this virus is now the leading cause of liver transplantation in the western world (3). Many patients treated with IFN-␣ alone or with a combination of IFN-␣ plus ribavirin fail to show a sustained virologic response and currently have no other treatment option. Given the high prevalence of the infection, HCV has become the focus of intensive research. Originally cloned in 1989 (1), the viral RNA genome is now well characterized. The ϳ9600-nucleotide genome is of positive polarity that encodes a ϳ3000-amino acid polyprotein, which is the precursor of at least 10 mature viral proteins: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. C is the nucleocapsid protein that binds and encapsulates the viral RNA genome (4), E1 and E2 are the virion glycoproteins, and p7 is of unknown function (5). The NS2 to NS5B proteins inclusively are thought to comprise nonstructural proteins involved in replication and polyprotein processing (6). The individual proteins are processed from the polyprotein by a combination of host and viral proteases. Host signal peptidases are responsible for the cleavages between C, E1, E2, p7, and NS2. The cleavage between NS2 and NS3 is performed in an autoproteolytic manner by the metal-dependent protease NS2/3 (7, 8). The proteolytic release of NS4A, NS4B, NS5A, and NS5B is catalyzed by the multifunctional NS3 enzyme, which in conjunction with the mature NS4A cofactor mediates efficient processing (for a review, see Ref. 9). The large polyprotein open...

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“…Since Edwards' disclosure of ␣-keto heterocycles as effective protease inhibitors, a number of enzyme inhibitors have been described on the basis of analogous design principles (60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70). The inhibitors developed herein define additional features that may contribute independently to binding affinity which, when Abbreviation: FAAH, fatty acid amide hydrolase.…”

mentioning

confidence: 99%

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

Boger

Sato

Lerner

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

263

264

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The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features-an optimal C12-C8 chain length, -unsaturation introduction at the corresponding arachidonoyl ⌬ 8,9 ͞⌬ 11,12 and oleoyl ⌬ 9,10 location, and an ␣-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 10 2 -10 3 times more potent than the corresponding trifluoromethyl ketones.

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Peptidomimetic Inhibitors of the Human Cytomegalovirus Protease

Cited by 94 publications

References 42 publications

Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor

Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor

An NS3 Serine Protease Inhibitor Abrogates Replication of Subgenomic Hepatitis C Virus RNA

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

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