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.

Relationship of potency and resilience to drug resistant mutations for GW420867X revealed by crystal structures of inhibitor complexes for wild-type, Leu100Ile, Lys101Glu, and Tyr188Cys mutant HIV-1 reverse transcriptases.

The selection of drug resistant viruses is a major problem in efforts to combat HIV and AIDS, hence, new compounds are required. We report crystal structures of wild-type and mutant HIV-1 RT with bound non-nucleoside (NNRTI) GW420867X, aimed at investigating the basis for its high potency and improved drug resistance profile compared to the first-generation drug nevirapine. GW420867X occupies a smaller volume than many NNRTIs, yet accesses key regions of the binding pocket. GW420867X has few contacts with Tyr188, hence, explaining the small effect of mutating this residue on inhibitor-binding potency. In a mutated NNRTI pocket, GW420867X either remains in a similar position compared to wild-type (RT(Leu100Ile) and RT(Tyr188Cys)) or rearranges within the pocket (RT(Lys101Glu)). For RT(Leu100Ile), GW420867X does not shift position, in spite of forming different side-chain contacts. The small bulk of GW420867X allows adaptation to a mutated NNRTI binding site by repositioning or readjustment of side-chain contacts with only small reductions in binding affinity.

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 A, 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.

Structure-activity relationship studies of novel benzophenones leading to the discovery of a potent, next generation HIV nonnucleoside reverse transcriptase inhibitor.

Despite the progress of the past two decades, there is still considerable need for safe, efficacious drugs that target human immunodeficiency virus (HIV). This is particularly true for the growing number of patients infected with virus resistant to currently approved HIV drugs. Our high throughput screening effort identified a benzophenone template as a potential nonnucleoside reverse transcriptase inhibitor (NNRTI). This manuscript describes our extensive exploration of the benzophenone structure-activity relationships, which culminated in the identification of several compounds with very potent inhibition of both wild type and clinically relevant NNRTI-resistant mutant strains of HIV. These potent inhibitors include 70h (GW678248), which has in vitro antiviral assay IC(50) values of 0.5 nM against wild-type HIV, 1 nM against the K103N mutant associated with clinical resistance to efavirenz, and 0.7 nM against the Y181C mutant associated with clinical resistance to nevirapine. Compound 70h has also demonstrated relatively low clearance in intravenous pharmacokinetic studies in three species, and it is the active component of a drug candidate which has progressed to phase 2 clinical studies.

Progesterone receptor ligand binding pocket flexibility: crystal structures of the norethindrone and mometasone furoate complexes.

Although progesterone, the natural ligand of the progesterone receptor (PR), has a hydrogen atom at the 17alpha position, other potent steroid agonists such as norethindrone and mometasone furoate have larger substituents at this position that are accommodated by the PR ligand binding pocket. Crystallographic analysis of PR ligand binding domain complexes clearly demonstrated that these moieties were accommodated by local shifts of the protein main chain and by adoption of alternative side chain rotamer conformations of ligand-proximal amino acids. These conformational changes imparted a ligand-specific volume to the binding pocket, from 490 A3 in the metribolone complex to 520 A3 in the norethindrone complex, 565 A3 in the progesterone complex, and 730 A3 in the mometasone furoate complex. Despite these marked alterations in binding pocket volume, critical interactions essential for establishment of an active AF2 conformation were maintained.

Design of non-nucleoside inhibitors of HIV-1 reverse transcriptase with improved drug resistance properties. 2.

HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) are part of the combination therapy currently used to treat HIV infection. The features of a new NNRTI drug for HIV treatment must include selective potent activity against both wild-type virus as well as against mutant virus that have been selected by use of current antiretroviral treatment regimens. Based on analogy with known HIV-1 NNRTI inhibitors and modeling studies utilizing the X-ray crystal structure of inhibitors bound in the HIV-1 RT, a series of substituted 2-quinolones was synthesized and evaluated as HIV-1 inhibitors.

Novel benzophenones as non-nucleoside reverse transcriptase inhibitors of HIV-1.

GW4511, GW4751, and GW3011 showed IC50 values < or =2 nM against wild type HIV-1 and <10 nM against 16 mutants. They were particularly potent against NNRTI-resistant viruses containing Y181C-, K103N-, and K103N-based double mutations, which account for a significant proportion of the clinical failure of the three currently marketed NNRTIs. The antiviral data together with the favorable pharmacokinetic data of GW4511 suggested that these benzophenones possess attributes of a new NNRTI drug candidate.

Structural basis for the improved drug resistance profile of new generation benzophenone non-nucleoside HIV-1 reverse transcriptase inhibitors.

Owing to the emergence of resistant virus, next generation non-nucleoside HIV reverse transcriptase inhibitors (NNRTIs) with improved drug resistance profiles have been developed to treat HIV infection. Crystal structures of HIV-1 RT complexed with benzophenones optimized for inhibition of HIV mutants that were resistant to the prototype benzophenone GF128590 indicate factors contributing to the resilience of later compounds in the series (GW4511, GW678248). Meta-substituents on the benzophenone A-ring had the designed effect of inducing better contacts with the conserved W229 while reducing aromatic stacking interactions with the highly mutable Y181 side chain, which unexpectedly adopted a "down" position. Up to four main-chain hydrogen bonds to the inhibitor also appear significant in contributing to resilience. Structures of mutant RTs (K103N, V106A/Y181C) with benzophenones showed only small rearrangements of the NNRTIs relative to wild-type. Hence, adaptation to a mutated NNRTI pocket by inhibitor rearrangement appears less significant for benzophenones than other next-generation NNRTIs.

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.

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 > or = 10-fold increases in their 50% inhibitory concentrations (IC50s) for 85% and 98% of the 55 selected isolates, respectively, whereas GW678248 had a > or = 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.

Antiviral activity of GW678248, a novel benzophenone nonnucleoside reverse transcriptase inhibitor.

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 (IC(50)s) between 0.8 and 6.8 nM. In HeLa CD4 MAGI cell culture virus replication assays, GW678248 has an IC(50) of < or =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 IC(50) 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 alpha-1 acid glycoprotein increased the IC(50) 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.

Fourier transform ion cyclotron resonance MS reveals the presence of a water molecule in an enzyme transition-state analogue complex.

The structures of several powerful inhibitors of hydrolytic enzymes resemble that of the altered substrate in the transition state, except that a hydrogen atom replaces one substituent (typically the leaving group). To test the hypothesis that a water molecule might be present in the gap resulting from this replacement, we examined a transition-state analogue complex formed by Escherichia coli cytidine deaminase by Fourier transform ion cyclotron resonance MS in electrospray mode. Upon nebularization from aqueous solution under conditions (pH 5.6) where the enzyme is active, cytidine deaminase remains dimeric in the vapor phase. In the presence of inhibitor, the enzyme's exact mass can be used to infer the presence at each active site of zinc, 5-fluoro-3,4-dihydrouridine, and a single water molecule.