Cytomegalovirus Protease Research Articles

The crystal structure of the Epstein-Barr virus protease shows rearrangement of the processed C terminus.

Epstein-Barr virus (EBV) belongs to the gamma-herpesvirinae subfamily of the Herpesviridae. The protease domain of the assemblin protein of herpesviruses forms a monomer-dimer equilibrium in solution. The protease domain of EBV was expressed in Escherichia coli and its structure was solved by X-ray crystallography to 2.3A resolution after inhibition with diisopropyl-fluorophosphate (DFP). The overall structure confirms the conservation of the homodimer and its structure throughout the alpha, beta, and gamma-herpesvirinae. The substrate recognition could be modelled using information from the DFP binding, from a crystal contact, suggesting that the substrate forms an antiparallel beta-strand extending strand beta5, and from the comparison with the structure of a peptidomimetic inhibitor bound to cytomegalovirus protease. The long insert between beta-strands 1 and 2, which was disordered in the KSHV protease structure, was found to be ordered in the EBV protease and shows the same conformation as observed for proteases in the alpha and beta-herpesvirus families. In contrast to previous structures, the long loop located between beta-strands 5 and 6 is partially ordered, probably due to DFP inhibition and a crystal contact. It also contributes to substrate recognition. The protease shows a specific recognition of its own C terminus in a binding pocket involving residue Phe210 of the other monomer interacting across the dimer interface. This suggests conformational changes of the protease domain after its release from the assemblin precursor followed by burial of the new C terminus and a possible effect onto the monomer-dimer equilibrium. The importance of the processed C terminus was confirmed using a mutant protease carrying a C-terminal extension and a mutated release site, which shows different solution properties and a strongly reduced enzymatic activity.

Pyrrolidine‐5,5‐trans‐lactams as Novel Mechanism‐Based Inhibitors of Human Cytomegalovirus Protease. Part 3. Potency and Plasma Stability.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Pyrrolidine-5,5- trans-lactams as novel mechanism-based inhibitors of human cytomegalovirus protease. Part 3: potency and plasma stability

Mechanism-based inhibitors of HCMV protease, which are stable to human plasma (≥20 h) and have single-figure potency in the μM range against HCMV protease, have been developed based on the dansylproline α-methyl pyrrolidine-5,5- trans-lactam nucleus.

Recombinant full-length human cytomegalovirus protease has lower activity than recombinant processed protease domain in purified enzyme and cell-based assays

Herpesviruses encode a protease that is essential for virus replication. The protease undergoes cleavage to a processed form during capsid maturation. A recombinant 75 kDa form of the protease from human cytomegalovirus was purified and compared with the recombinant 29 kDa processed form. Modification with an active site titrant suggested that most of each recombinant protease preparation was active (66 and 86%, respectively). Protease activity was compared using a low-molecular weight peptide substrate and the native substrate, capsid assembly protein. In addition, a cell-based assay for both enzymes was developed in which the target sequence of the protease has been fused inframe into the herpes simplex virus VP16 molecule. Cleavage of the fusion protein by the protease releases the carboxyl terminal transactivation domain, resulting in a decrease in the ability of the fusion molecule to transactivate a target promoter linked to a reporter gene in mammalian cells. Results suggest that the 75 kDa form of the enzyme is significantly less active than the 29 kDa form by all criteria.

Synthesis of a Peptidomimetic HCMV Protease Inhibitor Library

The human cytomegalovirus (HCMV) protease catalyzes the maturational process of the herpes virus assembly protein and plays a key role during the manufacture of viral capsid, and so is an attractive target for potential anti-herpes-virus agents with novel structures and new mechanisms. In this work, a peptidomimetic skeleton was designed and a chemical library containing 32 compounds with different substitutions on the skeleton was prepared by the oxidation of a precursor library, which was constructed from four types of building blocks: 4 carboxylic acids, 2 amines, 2 aldehydes and 2 isocyanides, based on multicomponent condensation following liquid phase strategies. The syntheses of the key building block isocyanides are presented.

RNase P-mediated inhibition of cytomegalovirus protease expression and viral DNA encapsidation by oligonucleotide external guide sequences.

External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. In this study, DNA-based EGS molecules were chemically synthesized to target the mRNA coding for the protease of human cytomegalovirus (HCMV). The EGS molecules efficiently directed human RNase P to cleave the target mRNA sequence in vitro. When EGSs were exogenously administered into HCMV-infected human foreskin fibroblasts, a reduction of about 80-90% in the expression level of the protease and a reduction of about 300-fold in HCMV growth were observed in the cells that were treated with a functional EGS, but not in cells that were not treated with the EGS or with a "disabled" EGS carrying nucleotide mutations that precluded RNase P recognition. Moreover, packaging of the viral DNA genome into the capsid was blocked in the cells treated with the functional EGS. These results indicate that HCMV protease is essential for viral DNA encapsidation. Moreover, our study provides direct evidence that exogenous administration of a DNA-based EGS can be used as a therapeutic approach for inhibiting gene expression and replication of a human virus.

Design and synthesis of pyrrolidine-5,5-trans-lactams (5-oxohexahydropyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of human cytomegalovirus protease. 2. Potency and chirality.

The stereospecific synthesis of a series of alpha-methylpyrrolidine-5,5-trans-lactam inhibitors of human cytomegalovirus (HCMV) protease is described. Examination of the SAR in this series has defined the size and chirality of the alpha-substituent, optimized the acyl substituent on the lactam nitrogen, and defined the steric constraint of this functionality. The SAR of the functionality on the pyrrolidine nitrogen of the trans-lactam has been investigated, and this has led to the discovery of potent serine protease inhibitors that are highly selective for the viral enzyme over the mammalian enzymes elastase, thrombin, and acetylcholine esterase. The mechanism of action of our lead compounds has been established by mass spectrometry, and enzymatic degradation of HCMV deltaAla protease acylated with these inhibitors showed that Ser 132 is the active site nucleophile. The crystal structure of HCMV protease was obtained and used to model the conformationally restricted, chiral (S)-proline-alpha-methyl-5,5-trans-lactams into the active site groove of the enzyme, enabling us to direct and rationalize the SAR in this series. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the dansyl-(S)-proline alpha-methyl-5,5-trans-lactam template, which have low nanomolar activity against the viral enzyme.

Molecular mechanism for dimerization to regulate the catalytic activity of human cytomegalovirus protease.

Biochemical studies indicate that dimerization is required for the catalytic activity of herpesvirus proteases, whereas structural studies show a complete active site in each monomer, away from the dimer interface. Here we report kinetic, biophysical and crystallographic characterizations of structure-based mutants in the dimer interface of human cytomegalovirus (HCMV) protease. Such mutations can produce a 1,700-fold reduction in the kcat while having minimal effects on the K(m). Dimer stability is not affected by these mutations, suggesting that dimerization itself is insufficient for activity. There are large changes in monomer conformation and dimer organization of the apo S225Y mutant enzyme. However, binding of an activated peptidomimetic inhibitor induced a conformation remarkably similar to the wild type protease. Our studies suggest that appropriate dimer formation may be required to indirectly stabilize the protease oxyanion hole, revealing a novel mechanism for dimerization to regulate enzyme activity.

Investigating the role of histidine 157 in the catalytic activity of human cytomegalovirus protease.

Herpesvirus proteases belong to a new class of serine proteases and contain a novel Ser-His-His catalytic triad, while classical serine proteases have an acidic residue as the third member. To gain a better understanding of the molecular basis for the functional role of the third-member His residue, we have carried out structural and biochemical investigations of human cytomegalovirus (HCMV) protease that bears mutations of the His157 third member. Kinetic studies showed that all the mutants have reduced catalytic activity. Structural studies revealed that a solvent molecule is hydrogen-bonded to the His63 second member and Ser134 in the H157A mutant, partly rescuing the activity of this mutant. This is confirmed by our kinetic and structural observations on the S134A/H157A double mutant, which showed further reductions in the catalytic activity. The structure of the H157A mutant is also in complex with the PMSF inhibitor. The H157E mutant has the best catalytic activity among the mutants; its structure, however, showed conformational readjustments of the His63 and Ser132 residues. The Ser132-His63 diad of HCMV protease has similar activity as the diads in classical serine proteases, whereas the contribution of the His157 third member to the catalysis is much smaller. Finally, structural comparisons revealed the presence of two conserved structural water molecules at the bottom of the S(1) pocket, suggesting a possible new direction for the design of HCMV protease inhibitors.

ChemInform Abstract: Metal Mediated Protease Inhibition: Design and Synthesis of Inhibitors of the Human Cytomegalovirus (hCMV) Protease.

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Design and synthesis of pyrrolidine-5,5-trans-lactams (5-oxo-hexahydro-pyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of human cytomegalovirus protease. 1. The alpha-methyl-trans-lactam template.

Mechanism-based inhibitors of human cytomegalovirus (HCMV) protease have been designed based on the pyrrolidine-5,5-trans-lactam ring system. New routes to the beta-methyl-, desmethyl-, and alpha-methyl-pyrrolidine-5,5-trans-lactam templates have been developed from 2,4-diaminobutyric acid. ESI/MS studies have shown that these inhibitors can bind covalently and reversibly to the viral enzyme in a time-dependent manner by a mechanism which is consistent with acylation of HCMV deltaAla protease at the active site nucleophile Ser 132. SAR in this series of pyrrolidine-5, 5-trans-lactams has defined the relative stereochemisty of the methyl substituent adjacent to the lactam carbonyl, the functionality on the lactam nitrogen, and the mechanism of action of this novel series of serine protease inhibitors against the HCMV deltaAla protease. Activity decreases on moving from the alpha-methyl to the desmethyl to the beta-methyl series. This selectivity is the opposite of that observed for these templates against the elastase and thrombin enzymes. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the Cbz-protected alpha-methyl-5,5-trans-lactam template which have low micromolar activity against the viral enzyme.

Metal mediated protease inhibition: design and synthesis of inhibitors of the human cytomegalovirus (hCMV) protease

A versatile synthetic route to a novel series of bis-imidazolemethanes designed to inhibit the hCMV protease has been developed and a series of potential metal binding inhibitors has been identified. In selectivity assays, the compounds were highly specific for CMV protease and showed no inhibition (IC 50>100 μM) of other prototypical serine proteases such as trypsin, elastase, and chymotrypsin. Although the presence of free zinc ions was found to be an absolute requirement for the in vitro biological activity of this class of inhibitor, the potency of the inhibitors could not be improved beyond the micromolar level.

Cytonic acids A and B: novel tridepside inhibitors of hCMV protease from the endophytic fungus Cytonaema species.

Two novel human cytomegalovirus protease inhibitors, cytonic acids A (1) and B (2), have been isolated from the solid-state fermentation of the endophytic fungi Cytonaema sp. Their structures as p-tridepside isomers were elucidated by MS and NMR methods.

A rational approach towards successful crystallization and crystal treatment of human cytomegalovirus protease and its inhibitor complex.

The crystallization and subsequent crystal treatment of both free human cytomegalovirus (hCMV) protease and its inhibitor complexes are reported. For free-enzyme crystals, diffraction was greatly improved by optimizing the crystallization conditions, raising the precipitant concentration in the reservoir and soaking the crystals in artificial mother liquors. Each of the six components in the final crystallization formula (16% PEG 4000, 0.1 M MES pH 6.0, 0.4 M LiCl, 10% glycerol, 5% t-butanol and 5 mM Na(2)S(2)O(3)) plays a distinctive role and is indispensable. A synergistic effect of Na(2)SO(4) and t--butanol on diffraction was observed and studied. A 2.0 A multiwavelength anomalous diffraction (MAD) data set was collected using a synchrotron-radiation source, leading to the elucidation of the three-dimensional structure of the enzyme. For the inhibitor-complex crystals, initial attempts with co-crystallization and soaking experiments at pH 6.0 did not produce conclusive results. Subsequently, experiments were designed to co-crystallize the complex at pH 8.0, the optimal pH for the enzyme and the inhibitor activity. Using 20-50 mM spermine in the crystallization buffer, crystals of two peptidomimetic inhibitor complexes were obtained at pH 7.5 and 8.0. Spermine was required for the inhibitor complexes to be crystallized at pH 8.0, possibly neutralizing net negative charges of hCMV protease owing to its acidic pI of 5.5. A 2.7 A data set was collected from one of the inhibitor complexes and the structure was determined using the molecular-replacement method.

The inhibition of human cytomegalovirus (hCMV) protease by hydroxylamine derivatives

Aryl hydroxylamine derivatives have been synthesised that are some of the most potent inhibitors of hCMV protease prepared to date (IC 50 14–60 nM). Mass spectrometry studies indicate that oxazinone derived hydroxylamines inhibit the enzyme by acylation of Ser 132 whereas non-oxazinone derived hydroxylamines appear to inhibit via formation of a sulfinanilide at Cys 138.

Synthesis and antiviral activity of monobactams inhibiting the human cytomegalovirus protease

A series of monobactam inhibitors of HCMV (No) protease bearing a heterocycle linked by a methylene group at C-4 is described. Inhibitors containing a heterocycle such as a 2-furyl, 2-thiophenyl, 4-methyl-2-tetrazole and 2-benzothiazole were found to be active in a plaque reduction assay. Furthermore, 2-benzothiazole derivatives were shown to inhibit the HCMV protease activity inside cells by using a cell transfection assay, indicating that their antiviral activity in the plaque reduction assay could be attributed to protease inhibition.

Activity in Monomers of Human Cytomegalovirus Protease

Herpesvirus proteases require dimerization for activity, although crystallographic data indicate that each monomeric subunit possesses a well-separated and complete active site. This suggests that dimerization stabilizes the monomeric protease subunits in an active conformation. Chemical cross-linking with disuccinimidyl glutarate was used to capture human cytomegalovirus protease in its various conformations. The cross-linked protease retained activity under mildly chaotropic conditions (0.25 to 0.75 M urea) in contrast to non-cross-linked protease which lost activity. Identification of active protease species by incorporation of radioactive diisopropylfluorophosphate showed that in addition to cross-linked dimers, cross-linked protease monomers were responsible for a significant fraction of the total protease activity. These results are consistent with the hypothesis that herpesvirus protease activation occurs by stabilization of an active conformer in the dimer.

Viral Protease Assay Based on GAL4 Inactivation Is Applicable to High-Throughput Screening in Mammalian Cells

We present an assay for viral proteases that relies on the proteolytic cleavage of substrate leading to the dissociation of the yeast transcription factor GAL4. A consensus substrate for the cytomegalovirus protease is fused between the DNA binding and transactivating domains of GAL4. Proteolysis inactivates the transcription factor which drives a luciferase reporter system. The assay is performed in mammalian cells, has a robust signal-to-noise ratio, and assesses proteolysis in a physiologic context. A unique feature of the assay is its ability to detect inhibitors of viral replication that act on viral targets other than the protease.

Characterization of the Human Cytomegalovirus Protease As an Induced-Fit Serine Protease and the Implications to the Design of Mechanism-Based Inhibitors

The conformational properties of the N-tert-butylacetyl-l-tert-butylglycyl-l-Nδ,Nδ-dimethylasparagyl-l-alanyl methyl ketone (MK) 1 and its terminal N-isopropylacetyl analogue 2 were investigated. Whereas these compounds are weak (mM IC50 range) inhibitors of the human cytomegalovirus (HCMV) protease, their activated carbonyl analogues are >1000-fold more potent (e.g., trifluoromethyl ketone 3, IC50 = 1.1 μM). A combination of NMR techniques demonstrated that MK 2 exists in solution as a relatively rigid and extended peptide structure and that the bulky side chains, notably the P3 tert-butyl group, greatly contribute to maintaining this solution conformation. Furthermore, transferred nuclear Overhauser effect (TRNOE) studies provided an enzyme-bound conformation of MK 2 that was found to be similar to its free solution structure and compares very well to the X-ray crystallographic structure of a related peptidyl inhibitor complexed to the enzyme. The fact that ligands such as MK 2 exist in solution in the bioactive conformation accounts, in part, for the observed inhibitory activity of activated ketone inhibitors bearing comparable peptidyl sequences. Comparison of the X-ray structures of HCMV protease apoenzyme and that of its complex with a related peptidyl α-ketoamide inhibitor allowed for a detailed analysis of the previously reported conformational change of the enzyme upon complexation of inhibitors such as 1 and 3. The above observations indicate that HCMV protease is a novel example of a serine protease that operates by an induced-fit mechanism for which complexation of peptidyl ligands results in structural changes which bring the enzyme to a catalytically active (or optimized) form. Kinetic and fluorescence studies are also consistent with an induced-fit mechanism in which a considerable proportion of the intrinsic ligand-binding energy is used to carry out the conformational reorganization of the protease. Issues related to the rational design of both mechanism- and nonmechanism-based inhibitors of HCMV protease, notably in light of the peptidyl ligand-induced optimization of its catalytic functioning, are discussed.

Inhibition of Human Cytomegalovirus Protease by Monocyclic β-Lactam Derivatives: Kinetic Characterization Using a Fluorescent Probe

Recent reports have demonstrated the potential of monocyclic β-lactam derivatives as inhibitors of human cytomegalovirus (HCMV) protease. Investigation of the mechanism of inhibition by NMR and mass spectrometry has revealed the presence of an acylenzyme intermediate suggesting that β-lactams are hydrolyzed by the enzyme and cause inhibition by competing with substrate. The potential of a fluorogenic β-lactam derivative for convenient kinetic characterization of this mechanism has been evaluated using 4S-(4-methylumbelliferone)-3R-methylazetidin-2-one-1-carboxylic acid (4-methylpyridyl) amide (1). Upon acylation of the enzyme, the fluorescent umbelliferone moiety is released, allowing for continuous monitoring of the hydrolytic process. Examination of a series of progress curves by numerical analysis has provided valuable information on acylation and deacylation rates which relate to the IC50 values observed for β-lactams. More importantly the potential of compound 1 as an active site titrating agent for ...