The virally encoded NS5B RNA-dependent RNA polymerase has emerged as a prime target in the search for specific HCV antivirals. A series of benzimidazole 5-carboxamide compounds inhibit the cellular RNA replication of a HCV subgenomic replicon and we have advanced our understanding of this class of inhibitors through a combination of complementary approaches that include biochemical cross-linking experiments with a photoreactive analogue followed by mass spectrometry analysis of the enzyme. A novel binding site has been localized for these inhibitors at the junction of the thumb domain and the N-terminal finger loop. Furthermore, the isolation and characterization of resistant replicon mutants that co-localize to this region distinguished this class of compounds from other non-nucleoside NS5B inhibitors that bind to distinct allosteric sites. Resistant mutations that emerged with the benzim- More than 2% of the world population are chronically infected with hepatitis C virus (HCV), 2 a flavivirus that is the etiological agent of non-A non-B hepatitis (1, 2). A large proportion of patients fail to achieve a sustained response to current therapies consisting of a combination of pegylated interferon and ribavirin. The discovery and development of specific anti-HCV chemotherapies aims to address this unmet clinical need and has focused on inhibitors of virally encoded functions. HCV encodes a linear polyprotein of ϳ3010 amino acids that is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins (for review, see Ref.3). One of the non-structural proteins, NS5B, catalyzes the RNA-dependent RNA polymerization of a negative strand intermediate and the subsequent generation of multiple copies of the plus strand viral genome; this enzyme has emerged as a principal target for chemotherapeutic inhibition of HCV replication (4).The three-dimensional structure of the NS5B polymerase reveals an organization comparable with other nucleic acid polymerases with the familiar features of fingers, palm, and thumb domains that are organized in a "right-hand" motif (5-7). A distinct feature of the HCV polymerase (and closely related RNA-dependent RNA polymerase) active site cavity is the protrusion of a unique -hairpin from the thumb subdomain that apparently plays a role in the initiation of de novo RNA synthesis as demonstrated by both structural and biochemical studies (8 -11). Another additional feature of the HCV polymerase is two loops that bridge the fingers and thumb subdomain and result in an encircled active site. This feature is now known to be shared by other RNA-dependent RNA polymerase from rhinovirus, bacteriophage 6, rabbit hemorrhagic disease virus, bovine viral diarrhea virus, Norwalk virus,. Interestingly, the interface between the HCV polymerase N-terminal 1 loop and the thumb subdomain is the location of a GTP binding site (8), although its precise biological role is unsolved.A number of different HCV polymerase inhibitors have emerged that can be broadly di...
The E6 Zn(2+)-binding protein of high-risk human papillomaviruses (HPVs) is one of the major transforming proteins encoded by these tumor viruses. A bacterial system was used to express wild type and truncated forms of HPV-16 E6 linked to GST. The recombinant proteins were released from GST through cleavage of a factor Xa site. Functional analysis of these proteins demonstrated that amino acids 2--142 comprise the minimal domain of E6 required to promote the degradation of p53 in vitro in a rabbit reticulocyte lysate. This purified protein, E6(Delta 143--151), required a high salt concentration for maximum solubility, eluted as a monomer on gel filtration, and was shown to bind two Zn(2+) ions by atomic absorption analysis. An N-terminal subdomain of E6 (amino acids 2--77, E6-N) was similarly purified. Unlike E6(Delta 143--151), E6--N was very soluble in low-salt buffers and hence was highly amenable to biophysical characterization. E6-N was shown to bind one Zn(2+) ion by electrospray mass spectrometry and by atomic absorption analysis. UV--visible spectroscopic analysis of Co(2+)-substituted E6--N revealed that four cysteine residues coordinate the metal ion. Mutational studies of all the cysteine residues in E6--N substantiated a critical role for Cys 30, 33, 63, and 66 in Zn(2+) binding and in proper folding of the subdomain. Equilibrium sedimentation of E6-N demonstrated that it is a monomer, like E6(Delta 143--151), at low concentrations, but dimerization occurs at high concentrations (K(d) = 0.1 mM). Finally, circular dichroism studies revealed significant secondary structure for both E6(Delta 143--151) and E6--N. The results support a model of monomeric E6 possessing two functionally critical Zn(2+)-binding motifs.
Azacyclohexatriene-2-ylidene (1), the 2-isomer of pyridine (2), has been generated by one-electron reduction of the corresponding radical cation in neutralization−reionization mass spectrometric experiments. The experimental finding that this molecule is stable on the microsecond time scale is in agreement with results of quantum chemical calculations that indicate both 1 and its radical cation, 1•+ , correspond to minima on the C5H5N and C5H5N•+ potential energy surfaces. The calculations predict that 1 is less stable than pyridine, 2, by 50 and 49 kcal/mol (MP2/6-31G** and CASSCF-MP2/6-31G**, respectively) or 47 kcal/mol (B3LYP/6-31G**), whereas the radical cations 1•+ and 2•+ are much closer in energy. The ylid ion 1•+ is predicted to be 6 and 7 kcal/mol lower in energy than 2•+ at the MP2 and CASSCF-MP2/6-31G** levels, respectively, and 1 kcal/mol higher according to the hybrid density functional theory. Calculations also suggest that facile isomerization of the ions is prohibited by an energy barrier, amounting to 62 and 57 kcal/mol at MP2/6-31G** and B3LYP/6-31G**, respectively, relative to 1•+ , which is even larger than the 38 kcal/mol obtained at both levels of theory required for the neutral transformation. Despite the substantial impediments, isomerization of excited species is possible since the lowest dissociation channels lie even higher in energy but the experimental observations confirm that neither the ions or neutrals undergo particularly facile isomerization. Using known thermochemical data a value for ΔH f (1•+ ) = 237 ± 5 kcal/mol was obtained from the measured appearance energy, 10.14 eV, of the C5H5N•+ ion generated from methyl picolinate, which is completely consistent with the theoretical predictions of 237−242 kcal/mol derived from the calculated energy differences between the various species and the known heat of formation of 2.
COMMUNICATIONSIn conclusion, dyotropic p-lactone-y-lactone ring enlargement follows the nonconcerted mechanism B; the migratory aptitudes of the substituents are in the order z-donor>ndonor > o-donor substituent. Migration of z-donor (and possibly also of o-donor) substituents proceeds via intermediate Z-1, whose lifetime depends on carbenium stabilization. The migration of n-donor (oxygen) substituents involves a bridged oxonium species 2-3. In all rearrangements an inversion at C4 is observed without exception. The stereochemical course of the reaction is better clarified,['I and its preparative value lies in the stereocontrolled formation of three contiguous stereogenic centers in the y-lactones 2c,e-h.
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