Human cytomegalovirus (hCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns. hCMV protease is essential for the production of mature infectious virions, as it performs proteolytic processing near the carboxy terminus (M-site) of the viral assembly protein precursor. hCMV protease is a serine protease, although it has little homology to other clans of serine proteases. Here we report the crystal structure of hCMV protease at 2.0 angstroms resolution, and show that it possesses a new polypeptide backbone fold. Ser 132 and His 63 are found in close proximity in the active site, confirming earlier biochemical and mutagenesis studies. The structure suggests that the third member of the triad is probably His 157. A dimer of the protease with an extensive interface is found in the crystal structure. This structure information will help in the design and optimization of inhibitors against herpesvirus proteases.
Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5 and 3 ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5 cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.
Human cytomegalovirus (HCMV) protease belongs to a new class of serine proteases, with a unique polypeptide backbone fold. The crystal structure of the protease in complex with a peptidomimetic inhibitor (based on the natural substrates and covering the P4 to P1' positions) has been determined at 2.7 A resolution. The inhibitor is bound in an extended conformation, forming an anti-parallel beta-sheet with the protease. The P3 and P1 side chains are less accessible to solvent, whereas the P4 and P2 side chains are more exposed. The inhibitor binding mode shows significant similarity to those observed for peptidomimetic inhibitors or substrates of other classes of serine proteases (chymotrypsin and subtilisin). HCMV protease therefore represents example of convergent evolution. In addition, large conformational differences relative to the structure of the free enzyme are observed, which may be important for inhibitor binding.
Interaction between the E2 protein and E1 helicase of human papillomaviruses (HPVs) is essential for the initiation of viral DNA replication. We recently described a series of small molecules that bind to the N-terminal transactivation domain (TAD) of HPV type 11 E2 and inhibits its interaction with E1 in vitro and in cellular assays. Here we report the crystal structures of both the HPV11 TAD and of a complex between this domain and an inhibitor, at 2.5-and 2.4-Å resolution, respectively. The HPV11 TAD structure is very similar to that of the analogous domain of HPV16. Inhibitor binding caused no significant alteration of the protein backbone, but movements of several amino acid side chains at the binding site, in particular those of Tyr-19, His-32, Leu-94, and Glu-100, resulted in the formation of a deep hydrophobic pocket that accommodates the indandione moiety of the inhibitor. Mutational analysis provides functional evidence for specific interactions between Tyr-19 and E1 and between His-32 and the inhibitor. A second inhibitor molecule is also present at the binding pocket. Although evidence is presented that this second molecule makes only weak interactions with the protein and is likely an artifact of crystallization, its presence defines additional regions of the binding pocket that could be exploited to design more potent inhibitors.Human papillomaviruses (HPVs) 1 are the etiological agents of malignant and benign lesions of the differentiating squamous or mucosal epithelium, notably of cervical cancer. Approximately 25 HPV types replicate in mucosal tissues of the anogenital tract. HPV16, -18, and -31 are the most prevalent "high-risk" types found in pre-cancerous or malignant lesions of the cervix. HPV6 and -11 are the most common "low-risk" types, which cause benign genital warts (condyloma acuminata), a less serious condition but one of the most common sexually transmitted diseases (1). Currently, no specific antivirals are available for the treatment of HPV infections.The small circular double-stranded DNA genome of papillomavirus is actively maintained as a multicopy episome in the nucleus of infected epithelial cells. This process is dependent on replication of the viral genome by the viral E1 and E2 proteins, in conjunction with the host DNA replication machinery. E2 is a sequence-specific DNA-binding protein that has a number of functions in the viral lifecycle. In addition to its role in the initiation of viral DNA replication, E2 is involved in regulating the transcription of viral genes (2-7), and in the segregation of the viral genome during cell division (8, 9). As a replication initiation factor, E2 binds with high affinity to specific sites located within the viral origin (ori) to help recruit it to the E1 helicase (10 -13). Formation of a ternary complex between E1, E2, and the origin relies not only on the interaction of E1 and E2 with specific DNA sequences at the origin but is also critically dependent on a direct interaction between these two proteins (14 -18).The 40-kDa E2 prote...
The in vitro resistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335.T he hepatitis C virus (HCV)-encoded NS3 protease is essential for viral replication and has long been considered an attractive target in drug design efforts (3, 5). NS3 protease inhibitors (PIs) can induce substantial reductions in HCV RNA plasma levels, and several candidates have progressed through clinical development to offer improved treatment options (for a review, see reference 27). Two PIs, boceprevir and telaprevir, were recently approved for use in combination with pegylated interferon (Peg-IFN) and ribavirin (1,6,7,19). The selection of drug-resistant variants is commonly observed in patients experiencing virologic rebound during treatment with PIs (16,[20][21][22]24).BI 201335 is a potent HCV NS3/4A PI (15, 28) currently in phase 3 clinical trials in combination with Peg-IFN and ribavirin as well as phase 2 assessment with other HCV direct acting antivirals in IFN-sparing regimens. BI 201335 exhibited a profound reduction in viral load when administered for 14 days as monotherapy in treatment-naïve patients or for 28 days in combination with Peg-IFN and ribavirin in treatment-experienced patients (16). In these studies, viral breakthrough was observed in most patients on monotherapy, whereas breakthrough was less frequent in patients undergoing combination treatment. Distinct resistant NS3 variants R155K and D168V predominated for genotype 1a and 1b (GT 1a and GT 1b), respectively (8,16).This study was designed to evaluate the genotypic and phenotypic profiles of the resistant variants that emerged during in vitro selection in the presence of BI 201335 in the replicon system and to relate these results to clinical observations. Replicons resistant to BI 201335 were selected in GT 1a H77 and GT 1b CON-1 replicon cell lines in the presence of 2 concentrations (100ϫ and 1,000ϫ drug concentration required to reduce HCV RNA or the luciferase reporter levels by 50% [EC 50 ]) of drug for 3 weeks and G-418 as previously described (9). With the lower concentration of BI 201335, resistant variants encoding NS3 changes at residues 155, 156, and 168 were selected with the GT 1b replicon, with D168G as the predominant variant (55%). R155K was the predominant variant (68%) selected with the GT 1a replicon (Table 1) and is consistent with the predominant variant selected in GT 1a HCV-infected patients (16). At the higher concentration of BI 201335, essentially only D168 variants were selected with D168 A and V as the predominant variants in both genotypes.In order to confirm that the mutations ob...
A series of N-tert-butylacetyl-l-tert-butylglycyl-l-Ngamma, Ngamma-dimethylasparagyl-l-alanyl-derived inhibitors (trifluoromethyl ketone 1, pentafluoroethyl ketone, 2, methyl ketone 3, and alpha-ketoamide 4, with respective KI values of 1.1, 0.1, 2100, and 0.2 microM) of the human cytomegalovirus protease were used to study the effect of binding of peptidyl inhibitors on the intrinsic fluorescence and CD properties of the enzyme. In the presence of saturating concentrations of compounds 1, 2, and 4, an identical blue shift in the fluorescence maximum of the enzyme upon specific tryptophan excitation was observed relative to that of the free protease. In the case of the methyl ketone 3, whose inhibition of the enzyme does not involve formation of a covalent adduct as evidenced by 13C NMR studies of carbonyl-labeled inhibitors, the blue shift in the emission was also observed. For both compounds 1 and 2 which exhibit slow-binding kinetics, the observed rate constants for the slow onset of inhibition of substrate hydrolysis correlate well with the kobs values of the time-dependent change in the emission spectra. Studies employing a double mutant of HCMV protease Ala143Gln/Trp42Phe identified Trp-42 as the principal fluorescence reporter. Taken together with information provided by our recent elucidation of the crystallographic structure of the enzyme [Tong, L., Qian, C., Massariol, M.-J., Bonneau, P. R., Cordingley, M. G., & Lagacé, L. (1996) Nature 383, 272], these observations are consistent with the inhibition of HCMV protease by peptidyl ketones involving a conformational change of the protease. A mechanism involving a kon limited by dehydration of the hydrated species, followed by rapid ligand binding and a conformational change prior to covalent adduct formation, is proposed for activated inhibitors such as 1 and 2.
A new polypeptide backbone fold for serine proteases has been identified based on the crystal structure ofhmnan cytomcgalovi.J.us protease. The structure was detem1ined at 2.5A resolution by the multiplewavelength anomalous diJ'fraction technique using the seleno-methionyl protein and refined at 2.0A resolution. It reveals a seven-stnmded mostly-antipm·allel 13-ban·eL which is sunounded by seven helices. The active site residues (Ser-132 and His-63) m·e situated on the outside of the 13-bmrel and in a groove on the surface of the protein. TI1e stmctme suggests that the third member of the catalytic triad is probably His-157. The protease of herpesviruses plays an essential role in the production of lllfectious virions 311d is an attractive tmget for the developement of antiherpes agents. The crystal stmcture information vvill help in the design m1d optimization of inhibitors agai.J.1st herpes virus protease.[1] Tong, L., Qian, C., Massariol, M. Human cytomegalovirus (HCMV) is a beta herpes vi.J.us. HCJ\.1Y, like all other members of the Herpes virus family, encodes a protease that is essential for capsid maturation and production of infectious virus. TI1e catalytic domain of the HCMV protease was produced in E.coli as a single-chain protein and was crystallized in space group C222 1 vvith two dimers per asymmetric unit. TI1e crystal stmcrure was deteffili.ned at 2.5A resolution using the ISAS method m1d noncryst:1llographico symmetry averaging. Om cmTent model has been refined against 2.3A data collected on an in1age plate at -170°C. The HCMV protease structure has a new fold different from that of any other 1.!1own protease. There is a central core comprising two orthogonal4-stranded beta sheets sunounded by eight alpha-helices. Residues in three flexible surface loops, including two associated with internal cleavage sites at ill11ino acids 143 and 209, have not been modeled into the wnent stmcture. Dimerization ofHCMV protease is mediated primarily by bmying four turns of alphaheli" (218-232) from one monomer into a pronounced depression in the smface of the other monomer. Only two of three residues previously in1plicated by ill11ino acid sequence alignment and mutagenesis asparticipating directly in catalysis (Ser-132 and His-63 but not Glu-122) me actually located in the active site. This novel structure is being used to finther understand the catalytic mechllilism. and to design inhibitors as potential anti-viral agents. Cysteine proteases related to mmnmalian inter!eukin-1~ convertlilg enzyme (ICE) and to its C. elegans homologue, CED-3, play a critical role in the biochemical events tl1at culminate in apoptosis. We have deteffili.ned the three-dimensional stmctme of a complex of tl1e hmnan CED-3 homologue CPP32/apopain witll a potent tetrapeptide-aldehyde inhibitor. I TI1e protein resembles ICE in overall structure, but its S 4 subsite is strikingly different in size and chemical composition. These differences account for the vmiation in specificity between tl1e ICE-and CED-3-related proteases and enable ...
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.
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