Kurt Weaver

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.

Dolutegravir (S/GSK1349572) Exhibits Significantly Slower Dissociation than Raltegravir and Elvitegravir from Wild-Type and Integrase Inhibitor-Resistant HIV-1 Integrase-DNA Complexes

ABSTRACT The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10−6 s−1 and a dissociative half-life (t1/2) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t1/2, at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

Crystallization of protein-ligand complexes.

Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals.

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.

Potent inhibitors of HIV-1 integrase display a two-step, slow-binding inhibition mechanism which is absent in a drug-resistant T66I/M154I mutant.

Two-metal binding HIV-1 integrase inhibitors (INIs) are potent inhibitors of HIV-1 in vitro and in patients. We report here for the first time the kinetics of inhibition of integrase-catalyzed strand transfer. First, the IC(50) values for each of six structurally distinct INIs decreased when a preincubation was included: S-1360 (1.3 microM vs 0.12 microM), L-731,988 (130 nM vs 9 nM), L-870,810 (130 nM vs 4 nM), raltegravir (300 nM vs 9 nM), elvitegravir (90 nM vs 6 nM), and GSK364735 (90 nM vs 6 nM). When reactions with these INIs were initiated with integrase, progress curve analyses indicated time-dependent inhibition, which could be fitted to a two-step mechanism of binding. Overall fitted K(i) values matched the IC(50) values measured with a preincubation: S-1360 (0.17 microM), L-731,988 (34 nM), L-870,810 (2.4 nM), raltegravir (10 nM), elvitegravir (4.0 nM), and GSK364735 (2.5 nM). To begin to understand the mechanism for this slow onset of inhibition and its possible impact on drug resistance, studies of resistance mutations were initiated. T66I/M154I exhibited little if any time-dependent inhibition by any of the six INIs, as measured by differences in potency upon preincubation or by progress curve analysis. These data demonstrate that slow binding is a signature of two-metal binding INIs, and that the second slow step is required for full potency. We discuss a possible structural explanation of the second slow step of inhibition and also the relationship between loss of time-dependent inhibition and drug resistance of this important new class of HIV-1 antiretroviral drugs.

Rational design of potent non-nucleoside inhibitors of HIV-1 reverse transcriptase.

A new series of non-nucleoside reverse transcriptase inhibitors based on an imidazole-amide biarylether scaffold has been identified and shown to possess potent antiviral activity against HIV-1, including the NNRTI-resistant Y188L mutated virus. X-ray crystallography of inhibitors bound to reverse transcriptase, including a structure of the Y188L RT protein, was used extensively to help identify and optimize the key hydrogen-bonding motif. This led directly to the design of compound 43 that exhibits remarkable antiviral activity (EC50<1 nM) against a wide range of NNRTI-resistant viruses and a favorable pharmacokinetic profile across multiple species.

Discovery of a Potent Boronic Acid Derived Inhibitor of the HCV RNA-Dependent RNA Polymerase.

A boronic acid moiety was found to be a critical pharmacophore for enhanced in vitro potency against wild-type hepatitis C replicons and known clinical polymorphic and resistant HCV mutant replicons. The synthesis, optimization, and structure-activity relationships associated with inhibition of HCV replication in a subgenomic replication system for a series of non-nucleoside boron-containing HCV RNA-dependent RNA polymerase (NS5B) inhibitors are described. A summary of the discovery of 3 (GSK5852), a molecule which entered clinical trials in subjects infected with HCV in 2011, is included.

Discovery of an inhibitor of insulin-like growth factor 1 receptor activation: Implications for cellular potency and selectivity over insulin receptor

Insulin-like growth factor 1 receptor (IGF-1R) is an attractive target for anti-cancer therapy due to its anti-apoptotic effect on tumor cells, but inhibition of insulin receptor (IR) may have undesired metabolic consequences. The primary sequences of the ATP substrate-binding sites of these receptors are identical and the crystal structures of the activated kinase domains are correspondingly similar. Thus, most small-molecule inhibitors described to date are equally potent against the activated kinase domains of IGF-1R and IR. In contrast, the non-phosphorylated kinase domains of these receptors have several structural features that may accommodate differences in binding affinity for kinase inhibitors. We used a cell-based assay measuring IGF-1R autophosphorylation as an inhibitor screen, and identified a potent purine derivative that is selective compared to IR. Surprisingly, the compound is a weak inhibitor of the activated IGF-1R tyrosine kinase domain. Biochemical and structural studies are presented that indicate the compound preferentially binds to the ATP site of non-phosphorylated IGF-1R compared to phosphorylated IGF-1R. The potential selectivity and potency advantages of this binding mode are discussed.

Monoclonal Antibody B9-2 Recognizes an 8 Amino Acid Sequence that Spans an Autocatalytic Cleavage Site Between the Two Subunits of Human Procaspase 8

Caspase 8 is synthesized in a zymogen form and must be proteolytically cleaved to be activated. The catalytically active enzyme is composed of two of the four cleavage products. We have determined that the monoclonal antibody B9-2 recognizes the octomer EMDLSSPQ. This sequence is shared by two of the cleavage products: a decamer linker region released after autocatalysis and the smaller subunit of the active enzyme.