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Eddy Arnold

Publications and source records attributed to Eddy Arnold.

At least 37 records · Page 2Linked to original sources
PubMed2005

Crystallography and the design of anti-AIDS drugs: conformational flexibility and positional adaptability are important in the design of non-nucleoside HIV-1 reverse transcriptase inhibitors.

Drug resistance is a key cause of failure for treatment of HIV infection. The efficacy of non-nucleoside reverse transcriptase inhibiting (NNRTI) drugs is impaired by rapid emergence of drug-resistance mutations. A multidisciplinary effort led to the discovery of the potent NNRTIs dapivirine and etravirine, both of which are diarylpyrimidine (DAPY) derivatives. Systematic structural and molecular modeling studies of HIV-1 reverse transcriptase (RT)/NNRTI complexes revealed different modes of inhibitor binding, and some of the DAPY inhibitors can bind to RT in different conformations. The torsional flexibility ("wiggling") of the inhibitors can generate numerous conformational variants and the compactness of the inhibitors permits significant repositioning and reorientation (translation and rotation) within the pocket ("jiggling"). Such adaptations appear to be critical for the ability of the diarylpyrimidine NNRTIs to retain their potency against a wide range of drug-resistant HIV-1 RTs. Exploitation of inhibitor conformational flexibility (such as torsional flexibility about strategically located chemical bonds) can be a powerful element of drug design, especially for the design of drugs that will be effective against rapidly mutating targets (which is a collection of related targets).

Acquired Immunodeficiency Syndrome↗
PubMed2004

Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

Amino Acid Sequence↗
PubMed2004

Roles of conformational and positional adaptability in structure-based design of TMC125-R165335 (etravirine) and related non-nucleoside reverse transcriptase inhibitors that are highly potent and effective against wild-type and drug-resistant HIV-1 variants.

Anti-AIDS drug candidate and non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC125-R165335 (etravirine) caused an initial drop in viral load similar to that observed with a five-drug combination in naïve patients and retains potency in patients infected with NNRTI-resistant HIV-1 variants. TMC125-R165335 and related anti-AIDS drug candidates can bind the enzyme RT in multiple conformations and thereby escape the effects of drug-resistance mutations. Structural studies showed that this inhibitor and other diarylpyrimidine (DAPY) analogues can adapt to changes in the NNRTI-binding pocket in several ways: (1). DAPY analogues can bind in at least two conformationally distinct modes; (2). within a given binding mode, torsional flexibility ("wiggling") of DAPY analogues permits access to numerous conformational variants; and (3). the compact design of the DAPY analogues permits significant repositioning and reorientation (translation and rotation) within the pocket ("jiggling"). Such adaptations appear to be critical for potency against wild-type and a wide range of drug-resistant mutant HIV-1 RTs. Exploitation of favorable components of inhibitor conformational flexibility (such as torsional flexibility about strategically located chemical bonds) can be a powerful drug design concept, especially for designing drugs that will be effective against rapidly mutating targets.

Anti-HIV Agents↗
PubMed2004

Structures of HIV-1 RT-DNA complexes before and after incorporation of the anti-AIDS drug tenofovir.

Tenofovir, also known as PMPA, R-9-(2-(phosphonomethoxypropyl)adenine, is a nucleotide reverse transcriptase (RT) inhibitor. We have determined the crystal structures of two related complexes of HIV-1 RT with template primer and tenofovir: (i) a ternary complex at a resolution of 3.0 A of RT crosslinked to a dideoxy-terminated DNA with tenofovir-diphosphate bound as the incoming substrate; and (ii) a RT-DNA complex at a resolution of 3.1 A with tenofovir at the 3' primer terminus. The tenofovir nucleotide in the tenofovir-terminated structure seems to adopt multiple conformations. Some nucleoside reverse transcriptase inhibitors, including 3TC and AZT, have elements ('handles') that project beyond the corresponding elements on normal dNTPs (the 'substrate envelope'). HIV-1 RT resistance mechanisms to AZT and 3TC take advantage of these handles; tenofovir's structure lacks handles that could protrude through the substrate envelope to cause resistance.

Adenine↗
PubMed2004

Crystal structure of RlmAI: implications for understanding the 23S rRNA G745/G748-methylation at the macrolide antibiotic-binding site.

The RlmA class of enzymes (RlmA(I) and RlmA(II)) catalyzes N1-methylation of a guanine base (G745 in Gram-negative and G748 in Gram-positive bacteria) of hairpin 35 of 23S rRNA. We have determined the crystal structure of Escherichia coli RlmA(I) at 2.8-A resolution, providing 3D structure information for the RlmA class of RNA methyltransferases. The dimeric protein structure exhibits features that provide new insights into its molecular function. Each RlmA(I) molecule has a Zn-binding domain, responsible for specific recognition and binding of its rRNA substrate, and a methyltransferase domain. The asymmetric RlmA(I) dimer observed in the crystal structure has a well defined W-shaped RNA-binding cleft. Two S-adenosyl-l-methionine substrate molecules are located at the two valleys of the W-shaped RNA-binding cleft. The unique shape of the RNA-binding cleft, different from that of known RNA-binding proteins, is highly specific and structurally complements the 3D structure of hairpin 35 of bacterial 23S rRNA. Apart from the hairpin 35, parts of hairpins 33 and 34 also interact with the RlmA(I) dimer.

Amino Acid Sequence↗
PubMed2004

Designing anti-AIDS drugs targeting the major mechanism of HIV-1 RT resistance to nucleoside analog drugs.

HIV reverse transcriptase (RT) is the target of a number of important anti-AIDS drugs. Drugs that inhibit RT are either nucleoside reverse transcriptase inhibitors (NRTIs) or non-nucleoside reverse transcriptase inhibitors (NNRTIs). Combinations of various anti-AIDS drugs (highly active anti-retroviral therapies or HAART) can reduce the viral load to non-detectable levels. However, the development of drug resistance leads to the emergence of HIV strains that are resistant to multiple anti-AIDS drugs. The nucleotide analogs that are used as anti-HIV-1 drugs lack the normal 3'-OH and as a consequence act as chain-terminators when incorporated into DNA. One mechanism of nucleoside analog resistance involves ATP-based excision to unblock chain-terminated primers and allow HIV replication to continue. There is an urgent need for new drugs and for new therapies that can overcome the excision mechanism of resistance. Compounds that disrupt the binding of the excision reaction substrate(s) (the blocked primer and/or ATP and/or pyrophosphate), or mimic the dinucleoside tetraphosphate product of the ATP-based excision reaction are potential inhibitors of excision. Detailed understanding of drug resistance mechanisms can reveal novel targets for anti-viral agents.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Taking aim at a moving target: designing drugs to inhibit drug-resistant HIV-1 reverse transcriptases.

HIV undergoes rapid genetic variation; this variation is caused primarily by the enormous number of viruses produced daily in an infected individual. Because of this variation, HIV presents a moving target for drug and vaccine development. The variation within individuals has led to the generation of diverse HIV-1 subtypes, which further complicates the development of effective drugs and vaccines. In general, it is more difficult to hit a moving target than a stationary target. Two broad strategies for hitting a moving target (in this case, HIV replication) are to understand the movement and to aim at the portions that move the least. In the case of anti-HIV drug development, the first option can be addressed by understanding the mechanism(s) of drug resistance and developing drugs that effectively inhibit mutant viruses. The second can be addressed by designing drugs that interact with portions of the viral machinery that are evolutionarily conserved, such as enzyme active sites.

Binding Sites↗
PubMed2004

Crystal structures of arginine deiminase with covalent reaction intermediates; implications for catalytic mechanism.

Arginine deiminase (ADI), an enzyme that hydrolyzes arginine to generate energy in many parasitic microorganisms, has potent anticancer activities and can halt growth of solid tumors. We determined the crystal structure of ADI from Mycoplasma arginini in two different forms (1.6 and 2.0 A resolution) using multiple isomorphous replacement. ADI shares common structural features with the arginine-catabolizing enzymes Arg:Gly amidinotransferase and dimethylarginine dimethyl-aminohydrolase; ADI contains an additional domain of five helices. The scissile C-N bonds of the substrates and the catalytic triads (Cys398-His269-Glu213 of ADI) for the three enzymes superimpose on each other. The ADI structure from form I crystals corresponds to a tetrahedral intermediate with four heteroatoms (1S, 2N, 1O) covalently bonded to the reaction-center carbon. The structure from form II crystals represents an amidino-enzyme complex; the reaction-center carbon is covalently bonded to Cys398 sulfur and two nitrogens, and the reacting water molecule is only 2.54 A away.

Amino Acid Sequence↗
PubMed2004

Effects of mutations in the G tract of the human immunodeficiency virus type 1 polypurine tract on virus replication and RNase H cleavage.

The RNase H cleavages that generate and remove the polypurine tract (PPT) primer during retroviral reverse transcription must be specific in order to create a linear viral DNA that is suitable for integration. Lentiviruses contain a highly conserved sequence consisting of six guanine residues at the 3' end of the PPT (hereafter referred to as the G tract). We introduced mutations into the G tract of a human immunodeficiency virus type 1-based vector and determined the effects on the virus titer and RNase H cleavage specificity. Most mutations in the G tract had little or no effect on the virus titer. Mutations at the second and fifth positions of the G tract increased the proportion of two-long-terminal-repeat (2-LTR) circle junctions with one or two nucleotide insertions. The second and fifth positions of the G tract make specific contacts with amino acids in the RNase H domain that are important for RNase H cleavage specificity. These complementary data define protein-nucleic acid interactions that help control the specificity of RNase H cleavage. When the G-tract mutants were analyzed in a viral background that was deficient in integrase, in most cases the proportion of consensus 2-LTR circle junctions increased. However, in the case of a mutant with Ts at the second and fifth positions of the G tract, the proportion of 2-LTR circle junctions containing the one-nucleotide insertion increased, suggesting that linear viral DNAs containing an extra base are substrates for integration. This result is consistent with the idea that the 3' end-processing reactions of retroviral integrases may help to generate defined ends from a heterogenous population of linear viral DNAs.

Base Sequence↗
PubMed2004

Correlations between factors determining the pharmacokinetics and antiviral activity of HIV-1 non-nucleoside reverse transcriptase inhibitors of the diaryltriazine and diarylpyrimidine classes of compounds.

OBJECTIVE: To investigate the important factors that determine the bioavailability and the antiviral activity of the diaryltriazine (DATA) and diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1 in animal species and humans using cell-based assays, physicochemical and computed parameters. METHODS: This naturalistic study included 15 parameters ranging from molecular mechanics calculations to phase I clinical trials. The calculated parameters were solvent-accessible surface area (SASA), polar surface area and Gibbs free energy of solvation. Physicochemical parameters comprised lipophilicity (octanol/water partition coefficient [cLogP]), ionisation constant (pKa), solubility and aggregate radius. Cell-based assays included human colonic adenocarcinoma cell (Caco-2) permeability (transepithelial transport), drug metabolism and antiviral activity (negative logarithm of the molar effective concentration inhibiting viral replication by 50% [pEC50]). Exposure was tested in rats, dogs and human volunteers. RESULTS: Of the 15 parameters, eight correlated consistently among one another. Exposure (area under the plasma concentration-time curve [AUC]) in humans correlated positively with that in rats (r = 1.00), with transepithelial transport (r = 0.83), lipophilicity (r = 0.60), ionisability (r = 0.89), hydrodynamic radius of aggregates (r = 0.66) and with antiviral activity (r = 0.61). Exposure in humans was also seen to correlate negatively with SASA (r = -0.89). No consistent correlation was found between exposure in dogs and the eight parameters. Of the 14 DATA/DAPY molecules, 11 form aggregates with radii between 34 and 100 nm. CONCLUSIONS: We observed correlations between exposure in humans with exposure in rats, transepithelial transport (Caco-2 cells), ionisability, lipophilicity, aggregate radius and SASA in the class of DATA/DAPY NNRTI compounds. The lipophilic DATA/DAPY compounds form aggregates. It can be assumed that absorption in the intestinal tract and endocytosis in infected cells of these lipophilic compounds are governed by the common phenomenon of aggregate formation. As the lymphatic system offers a pathway for intestinal uptake of aggregates, this may offer a therapeutic advantage in the treatment of HIV-1 infection. Although it was not the objective of the study, we found that the rat was a better in vivo model than the dog for the prediction of systemic exposure in this particular set of compounds.

Animals↗
PubMed2003

Molecular model of SARS coronavirus polymerase: implications for biochemical functions and drug design.

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV plays a pivotal role in viral replication and is a potential target for anti-SARS therapy. There is a lack of structural or biochemical data on any coronavirus polymerase. To provide insights into the structure and function of SARS-CoV RdRp, we have located its conserved motifs that are shared by all RdRps, and built a three-dimensional model of the catalytic domain. The structural model permits us to discuss the potential functional roles of the conserved motifs and residues in replication and their potential interactions with inhibitors of related enzymes. We predict important structural attributes of potential anti-SARS-CoV RdRp nucleotide analog inhibitors: hydrogen-bonding capability for the 2' and 3' groups of the sugar ring and C3' endo sugar puckering, and the absence of a hydrophobic binding pocket for non-nucleoside analog inhibitors similar to those observed in hepatitis C virus RdRp and human immunodeficiency virus type 1 reverse transcriptase. We propose that the clinically observed resistance of SARS to ribavirin is probably due to perturbation of the conserved motif A that controls rNTP binding and fidelity of polymerization. Our results suggest that designing anti-SARS therapies can benefit from successful experiences in design of other antiviral drugs. This work should also provide guidance for future biochemical experiments.

Amino Acid Motifs↗
PubMed2003

Crystal structure of human tryptophanyl-tRNA synthetase catalytic fragment: insights into substrate recognition, tRNA binding, and angiogenesis activity.

Human tryptophanyl-tRNA synthetase (hTrpRS) produces a full-length and three N terminus-truncated forms through alternative splicing and proteolysis. The shortest fragment that contains the aminoacylation catalytic fragment (T2-hTrpRS) exhibits the most potent angiostatic activity. We report here the crystal structure of T2-hTrpRS at 2.5 A resolution, which was solved using the multi-wavelength anomalous diffraction method. T2-hTrpRS shares a very low sequence homology of 22% with Bacillus stearothermophilus TrpRS (bTrpRS); however, their overall structures are strikingly similar. Structural comparison of T2-hTrpRS with bTrpRS reveals substantial structural differences in the substrate-binding pocket and at the entrance to the pocket that play important roles in substrate binding and tRNA binding. T2-hTrpRS has a wide opening to the active site and adopts a compact conformation similar to the closed conformation of bTrpRS. These results suggest that mammalian and bacterial TrpRSs might use different mechanisms to recognize the substrate. Modeling studies indicate that tRNA binds with the dimeric enzyme and interacts primarily with the connective polypeptide 1 of hTrpRS via its acceptor arm and the alpha-helical domain of hTrpRS via its anticodon loop. Our results also suggest that the angiostatic activity is likely located at the alpha-helical domain, which resembles the short chain cytokines.

Adenosine Triphosphate↗
PubMed2003

Trapping HIV-1 reverse transcriptase before and after translocation on DNA.

A disulfide cross-linking strategy was used to covalently trap as a stable complex (complex N) a short-lived, kinetic intermediate in DNA polymerization. This intermediate corresponds to the product of polymerization prior to translocation. We also prepared the trapped complex that corresponds to the product of polymerization after translocation (complex P). The cross-linking method that we used is a variation of a technique developed by the Verdine and Harrison laboratories. It involves disulfide interchange between an engineered sulfhydryl group of the protein (Q258C mutation) and a disulfide-containing tether attached at the N(2) amino group of a modified dG in either the template or the primer strand of the nucleic acid. We report here a highly efficient synthesis of the precursor, bis(3-aminopropyl)disulfide dihydrochloride, used to introduce this substituent into the oligonucleotide. Efficient cross-linking takes place when the base pair containing the substituent is positioned seven registers from the dNTP-binding site (N site) and the N site is occupied. Complex N, but not complex P, is a substrate for the ATP-based excision reaction that unblocks nucleoside reverse transcriptase inhibitor (NRTI)-terminated primers and causes resistance to several NRTIs, confirming predictions that the excision reaction takes place only when the 3'-end of the primer is bound at the N site. These techniques can be used for biochemical and structural studies of the mechanism of DNA polymerization, translocation, and excision-based resistance of RT to NRTIs. They may also be useful in studying other DNA or RNA polymerases or other enzymes.

Adenosine Triphosphate↗
PubMed2003

Crystal structure of D-Hydantoinase from Burkholderia pickettii at a resolution of 2.7 Angstroms: insights into the molecular basis of enzyme thermostability.

D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD(Bp)) at a 2.7-A resolution. The structure of HYD(Bp) consists of a core (alpha/beta)(8) triose phosphate isomerase barrel fold and a beta-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYD(Bp) shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYD(Tsp)) and Bacillus stearothermophilus (HYD(Bst)), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs. The more thermostable HYD(Tsp) contains more aromatic residues in the interior of the structure than HYD(Bp) and HYD(Bst). Changes of large aromatic residues in HYD(Tsp) to smaller residues in HYD(Bp) or HYD(Bst) decrease the hydrophobicity and create cavities inside the structure. HYD(Tsp) has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD(Bp) and HYD(Bst). Besides, HYD(Tsp) also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.

Amidohydrolases↗
PubMed2003

The hepatitis B virus polymerase mutation rtV173L is selected during lamivudine therapy and enhances viral replication in vitro.

Therapy of chronic hepatitis B virus (HBV) infection with the polymerase inhibitor lamivudine frequently is associated with the emergence of viral resistance. Genotypic changes in the YMDD motif (reverse transcriptase [rt] mutations rtM204V/I) conferred resistance to lamivudine as well as reducing the in vitro replication efficiency of HBV. A second mutation, rtL180M, was previously reported to partially restore replication fitness as well as to augment drug resistance in vitro. Here we report the functional characterization of a third polymerase mutation (rtV173L) associated with resistance to lamivudine and famciclovir. rtV173L was observed at baseline in 9 to 22% of patients who entered clinical trials of adefovir dipivoxil for the treatment of lamivudine-resistant HBV. In these patients, rtV173L was invariably found as a third mutation in conjunction with rtL180M and rtM204V. In vitro analyses indicated that rtV173L did not alter the sensitivity of wild-type or lamivudine-resistant HBV to lamivudine, penciclovir, or adefovir but instead enhanced viral replication efficiency. A molecular model of HBV polymerase indicated that residue rtV173 is located beneath the template strand of HBV nucleic acid near the active site of the reverse transcriptase. Substitution of leucine for valine at this residue may enhance polymerization either by repositioning the template strand of nucleic acid or by affecting other residues involved in the polymerization reaction. Together, these results suggest that rtV173L is a compensatory mutation that is selected in lamivudine-resistant patients due to an enhanced replication phenotype.

2-Aminopurine↗
PubMed2002

Structures of HIV-1 reverse transcriptase with pre- and post-translocation AZTMP-terminated DNA.

AZT (3'-azido-3'-deoxythymidine) resistance involves the enhanced excision of AZTMP from the end of the primer strand by HIV-1 reverse transcriptase. This reaction can occur when an AZTMP-terminated primer is bound at the nucleotide-binding site (pre-translocation complex N) but not at the 'priming' site (post-translocation complex P). We determined the crystal structures of N and P complexes at 3.0 and 3.1 A resolution. These structures provide insight into the structural basis of AZTMP excision and the mechanism of translocation. Docking of a dNTP in the P complex structure suggests steric crowding in forming a stable ternary complex that should increase the relative amount of the N complex, which is the substrate for excision. Structural differences between complexes N and P suggest that the conserved YMDD loop is involved in translocation, acting as a springboard that helps to propel the primer terminus from the N to the P site after dNMP incorporation.

DNA↗
PubMed2002

Crystal structure of a human rhinovirus that displays part of the HIV-1 V3 loop and induces neutralizing antibodies against HIV-1.

We report the 2.7 A resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I beta turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.

Crystallography, X-Ray↗