Reiterative in vitro selection-amplification from random oligonucleotide libraries allows the identification of molecules with specific functions such as binding to specific proteins. The therapeutic usefulness of such molecules depends on their high affinity and nuclease resistance. Libraries of RNA molecules containing 2'amino-(2'NH2)- or 2'fluoro-(2'F)-2'-deoxypyrimidines could yield ligands with similar nuclease resistance but not necessarily with similar affinities. This is because the intramolecular helices containing 2'NH2 have lower melting temperatures (Tm) compared with helices containing 2'F, giving them thermodynamically less stable structures and possibly weaker affinities. We tested these ideas by isolating high-affinity ligands to human keratinocyte growth factor from libraries containing modified RNA molecules with either 2'NH2 or 2'F pyrimidines. We demonstrated that 2'F RNA ligands have affinities (Kd approximately 0.3-3 pM) and bioactivities (Ki approximately 34 pM) superior to 2'NH2 ligands (Kd approximately 400 pM and Ki approximately 10 nM). In addition, 2'F ligands have extreme thermo-stabilities (Tm approximately 78 degrees C in low salt, and specificities).
The interaction between lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF) and human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for HIV-1 replication. Homogeneous time-resolved fluorescence resonance energy transfer assays were developed to characterize HIV-1 integrase dimerization and the interaction between LEDGF and IN dimers. Using these assays in an equilibrium end point dose-response format with mathematical modeling, we determined the dissociation constants of IN dimers (K dimer ؍ 67.8 pM) and of LEDGF from IN dimers (K d ؍ 10.9 nM). When used in a kinetic format, the assays allowed the determination of the on-and off-rate constants for these same interactions. Targeting the viral integration process with small molecules as a strategy to inhibit HIV-1 2 replication has recently yielded an important new class of antiviral drugs. One integrase strand transfer inhibitor (INSTI), raltegravir (MK-0518), has been approved by the Food and Drug Administration for treatment of HIV-1 infection, and a second drug, elvitegravir (GS-9137), is in late stage clinical development. Based on binding experiments (1) and molecular modeling (2), strand transfer inhibitors are thought to interact with a pocket in the active site of integrase that is formed after 3Ј-processing of the viral DNA ends. INSTIs thus prevent the integrase-viral DNA complex from engaging host target DNA. More recently, a second site on integrase that represents the binding site for the cellular cofactor LEDGF was demonstrated to be a viable and attractive target for antiviral drug discovery (3-5). A small molecule inhibitor targeting this second site may retain activity against viral mutants resistant to INSTIs and complement the antiviral activity of INSTIs, akin to non-nucleoside reverse transcriptase inhibitors with nucleoside reverse transcriptase inhibitors. Hence, compounds that interact with the LEDGF binding pocket on IN could be used in combination with INSTIs to decrease the likelihood of resistance emergence.The cellular cofactor LEDGF has been identified as the dominant binding partner of HIV-1 integrase in human cells (6 -10). LEDGF interacts with integrase primarily through an ϳ80-amino acid domain termed the integrase binding domain (IBD) (11). A solution structure of the IBD has been derived (12). In addition, several co-crystal structures involving IBD and IN domains have been solved and include the following: IBD bound to a dimer of HIV-1 integrase catalytic core domain (CCD), IBD bound to an HIV-2 two-domain integrase (N-terminal domain (NTD) and catalytic core domain), and IBD bound to a maedi-visna virus two-domain (NTD ϩ CCD) integrase (13-15). Several lines of evidence point to the requirement of LEDGF for HIV-1 replication as a viral cofactor as follows. 1) Mutations in integrase that preserve the catalytic activity of the enzyme but cause defects in viral replication also disrupt integrase interaction with LEDGF (16 -18). 2) Suppression of LEDGF expression mediated by smal...
Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC 50 ) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC 50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC 50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC 50 values equivalent to those for the wild type.
The hepatitis C virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-HCV compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (ϳ70% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh-7 cells. In this report, we describe the selection of highly permissive Huh-7 cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support the replication of multiple genotype 1a replicons (including the H77 and SF9 strains), are significantly more permissive to genotype 1a HCV replication than parental Huh7-Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing of the nonstructural region of 12 stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes.
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