Steven A. Short

Structural Basis for the Resilience of Efavirenz (DMP-266) to Drug Resistance Mutations in HIV-1 Reverse Transcriptase

BACKGROUND Efavirenz is a second-generation non-nucleoside inhibitor of HIV-1 reverse transcriptase (RT) that has recently been approved for use against HIV-1 infection. Compared with first-generation drugs such as nevirapine, efavirenz shows greater resilience to drug resistance mutations within HIV-1 RT. In order to understand the basis for this resilience at the molecular level and to help the design of further-improved anti-AIDS drugs, we have determined crystal structures of efavirenz and nevirapine with wild-type RT and the clinically important K103N mutant. RESULTS The relatively compact efavirenz molecule binds, as expected, within the non-nucleoside inhibitor binding pocket of RT. There are significant rearrangements of the drug binding site within the mutant RT compared with the wild-type enzyme. These changes, which lead to the repositioning of the inhibitor, are not seen in the interaction with the first-generation drug nevirapine. CONCLUSIONS The repositioning of efavirenz within the drug binding pocket of the mutant RT, together with conformational rearrangements in the protein, could represent a general mechanism whereby certain second-generation non-nucleoside inhibitors are able to reduce the effect of drug-resistance mutations on binding potency.

Product Release Is the Major Contributor tok cat for the Hepatitis C Virus Helicase-catalyzed Strand Separation of Short Duplex DNA

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3′ single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5′-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3′-fluorescein-tagged 21-mer (F21).k cat for this reaction was 0.12 s−1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs (∼2) were disrupted concertedly. The maximal value of the rate constant (k eff) describing the late phase of the reaction (strand separation) was 0.5 s−1, which was 4-fold greater than k cat. Release of HF31 from E·HF31 in the presence of ATP (0.21 s−1) was the major contributor tok cat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

Antiviral Activity of GW678248, a Novel Benzophenone Nonnucleoside Reverse Transcriptase Inhibitor

ABSTRACT The compound GW678248 is a novel benzophenone nonnucleoside reverse transcriptase inhibitor (NNRTI). Preclinical assessment of GW678248 indicates that this compound potently inhibits wild-type (WT) and mutant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in biochemical assays, with 50% inhibitory concentrations (IC50s) between 0.8 and 6.8 nM. In HeLa CD4 MAGI cell culture virus replication assays, GW678248 has an IC50 of ≤21 nM against HIV-1 isogenic strains with single or double mutations known to be associated with NNRTI resistance, including L100I, K101E, K103N, V106A/I/M, V108I, E138K, Y181C, Y188C, Y188L, G190A/E, P225H, and P236L and various combinations. An IC50 of 86 nM was obtained with a mutant virus having V106I, E138K, and P236L mutations that resulted from serial passage of WT virus in the presence of GW678248. The presence of 45 mg/ml human serum albumin plus 1 mg/ml α-1 acid glycoprotein increased the IC50 approximately sevenfold. Cytotoxicity studies with GW678248 indicate that the 50% cytotoxicity concentration is greater than the level of compound solubility and provides a selectivity index of >2,500-fold for WT, Y181C, or K103N HIV-1. This compound exhibits excellent preclinical antiviral properties and, as a prodrug designated GW695634, is being developed as a new generation of NNRTI for the treatment of HIV-1 in combination with other antiretroviral agents.

Structural insights into mechanisms of non-nucleoside drug resistance for HIV-1 reverse transcriptases mutated at codons 101 or 138

Lys101Glu is a drug resistance mutation in reverse transcriptase clinically observed in HIV‐1 from infected patients treated with the non‐nucleoside inhibitor (NNRTI) drugs nevirapine and efavirenz. In contrast to many NNRTI resistance mutations, Lys101(p66 subunit) is positioned at the surface of the NNRTI pocket where it interacts across the reverse transcriptase (RT) subunit interface with Glu138(p51 subunit). However, nevirapine contacts Lys101 and Glu138 only indirectly, via water molecules, thus the structural basis of drug resistance induced by Lys101Glu is unclear. We have determined crystal structures of RT(Glu138Lys) and RT(Lys101Glu) in complexes with nevirapine to 2.5 Å, allowing the determination of water structure within the NNRTI‐binding pocket, essential for an understanding of nevirapine binding. Both RT(Glu138Lys) and RT(Lys101Glu) have remarkably similar protein conformations to wild‐type RT, except for significant movement of the mutated side‐chains away from the NNRTI pocket induced by charge inversion. There are also small shifts in the position of nevirapine for both mutant structures which may influence ring stacking interactions with Tyr181. However, the reduction in hydrogen bonds in the drug‐water‐side‐chain network resulting from the mutated side‐chain movement appears to be the most significant contribution to nevirapine resistance for RT(Lys101Glu). The movement of Glu101 away from the NNRTI pocket can also explain the resistance of RT(Lys101Glu) to efavirenz but in this case is due to a loss of side‐chain contacts with the drug. RT(Lys101Glu) is thus a distinctive NNRTI resistance mutant in that it can give rise to both direct and indirect mechanisms of drug resistance, which are inhibitor‐dependent.

Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp(-) Escherichia coli.

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.

Preparative isolation of DNA and biologically active mRNA from diethylaminoethyl membrane

Abstract A simple method is described for the preparative isolation of both DNA and mRNA after electrophoretic separation in agarose gels. The method is based on the quantitative binding of the nucleic acid to a membrane containing charged diethylaminoethyl (DEAE) groups, followed by elution under high salt conditions. Fragments of bacteriophage λ cut with Hind III, ranging in size from 565 to 9536 bp were recovered at efficiencies ranging from 84% to 35%, respectively. Poly A + mRNAs from hen oviduct were recovered with an efficiency of 65–80%. Recovery of biological activity was about 70%.

Allosteric Mechanism of Induction of CytR-regulated Gene Expression CytR REPRESSOR-CYTIDINE INTERACTION

Transcription from cistrons of theEscherichia coli CytR regulon is activated by E. coli cAMP receptor protein (CRP) and repressed by a multiprotein complex composed of CRP and CytR. De-repression results when CytR binds cytidine. CytR is a homodimer and a LacI family member. A central question for all LacI family proteins concerns the allosteric mechanism that couples ligand binding to the protein-DNA and protein-protein interactions that regulate transcription. To explore this mechanism for CytR, we analyzed nucleoside binding in vitro and its coupling to cooperative CytR binding to operator DNA. Analysis of the thermodynamic linkage between sequential cytidine binding to dimeric CytR and cooperative binding of CytR to deoP2 indicates that de-repression results from just one of the two cytidine binding steps. To test this conclusion in vivo, CytR mutants that have wild-type repressor function but are cytidine induction-deficient (CID) were identified. Each has a substitution for Asp281or neighboring residue. CID CytR281N was found to bind cytidine with three orders of magnitude lower affinity than wild-type CytR. Other CytR mutants that do not exhibit the CID phenotype were found to bind cytidine with affinity similar to wild-type CytR. The rate of transcription regulated by heterodimeric CytR composed of one CytR281N and one wild-type subunit was compared with that regulated by wild-type CytR under inducing conditions. The data support the conclusion that the first cytidine binding step alone is sufficient to induce.

Anti-Human Immunodeficiency Virus Type 1 Activity of the Nonnucleoside Reverse Transcriptase Inhibitor GW678248 in Combination with Other Antiretrovirals against Clinical Isolate Viruses and In Vitro Selection for Resistance

ABSTRACT GW678248, a novel nonnucleoside reverse transcriptase inhibitor, has been evaluated for anti-human immunodeficiency virus activity in a variety of in vitro assays against laboratory strains and clinical isolates. When GW678248 was tested in combination with approved drugs in the nucleoside and nucleotide reverse transcriptase inhibitor classes or the protease inhibitor class, the antiviral activities were either synergistic or additive. When GW678248 was tested in combination with approved drugs in the nonnucleoside reverse transcriptase inhibitor class, the antiviral activities were either additive or slightly antagonistic. Clinical isolates from antiretroviral drug-experienced patients were selected for evaluation of sensitivity to GW678248 in a recombinant virus assay. Efavirenz (EFV) and nevirapine (NVP) had ≥10-fold increases in their 50% inhibitory concentrations (IC50s) for 85% and 98% of the 55 selected isolates, respectively, whereas GW678248 had a ≥10-fold increase in the IC50 for only 17% of these isolates. Thus, 81 to 83% of the EFV- and/or NVP-resistant viruses from this data set were susceptible to GW678248. Virus populations resistant to GW678248 were selected by in vitro dose-escalating serial passage. Resistant progeny viruses recovered after eight passages had amino acid substitutions V106I, E138K, and P236L in the reverse transcriptase-coding region in one passage series and amino acid substitutions K102E, V106A, and P236L in a second passage series.

The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twist

Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined alpha-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

2-amino-9-(3-azido-2,3-dideoxy-β-d-erythro-pentofuranosyl)-6-substituted-9H-purines: Synthesis and anti-HIV activity

A series of 2-amino-9-(3-azido-2,3-dideoxy-beta-D-erythro-pentofuranosyl)-6- substituted-9H-purines was synthesized and tested for the ability to protect MT4 cells from the cytopathic effect of HIV-1IIIB. These compounds were prepared by a combination of chemical and enzymatic reactions. Some of the nucleoside analogs with 6-alkoxy, 6-alkylamino, or 6-arylamino substituents were active against HIV-1IIIB. Their IC50 values were in the range of 2-60 microM. In contrast, analogs with 6-thio, 6-alkylthio, 6-methyl, or 6-carbonitrile substituents did not protect cells from the cytopathic effect of HIV infection.