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Biomedical subjects

D A Matthews

Publications and source records attributed to D A Matthews.

At least 19 recordsLinked to original sources
PubMed2005

Conservation of amino acids in human rhinovirus 3C protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor.

The picornavirus 3C protease is required for the majority of proteolytic cleavages that occur during the viral life cycle. Comparisons of published amino acid sequences from 6 human rhinoviruses (HRV) and 20 human enteroviruses (HEV) show considerable variability in the 3C protease-coding region but strict conservation of the catalytic triad residues. Rupintrivir (formerly AG7088) is an irreversible inhibitor of HRV 3C protease with potent in vitro activity against all HRV serotypes (48 of 48), HEV strains (4 of 4), and untyped HRV field isolates (46 of 46) tested. To better understand the relationship between in vitro antiviral activity and 3C protease-rupintrivir binding interactions, we performed nucleotide sequence analyses on an additional 21 HRV serotypes and 11 HRV clinical isolates. Antiviral activity was also determined for 23 HRV clinical isolates and four additional HEV strains. Sequence comparison of 3C proteases (n = 58) show that 13 and 11 of the 14 amino acids that are involved in side chain interactions with rupintrivir are strictly conserved among HRV and HEV, respectively. These sequence analyses are consistent with the comparable in vitro antiviral potencies of rupintrivir against all HRV serotypes, HRV isolates, and HEV strains tested (50% effective concentration range, 3 to 183 nM; n = 125). In summary, the conservation of critical amino acid residues in 3C protease and the observation of potent, broad-spectrum antipicornavirus activity of rupintrivir highlight the advantages of 3C protease as an antiviral target.

3C Viral Proteases↗
PubMed2004

Precursor of human adenovirus core polypeptide Mu targets the nucleolus and modulates the expression of E2 proteins.

We have examined the subcellular localization properties of human adenovirus 2 (HAdV-2) preMu and mature Mu (pX) proteins as fusions with enhanced green fluorescence protein (EGFP). We determined that preMu is exclusively a nucleolar protein with a single nucleolar accumulation signal within the Mu sequence. In addition, we noted that both preMu-EGFP and Mu-EGFP are excluded from adenovirus DNA-binding protein (DBP)-rich replication centres in adenovirus-infected cells. Surprisingly, we observed that cells in which preMu-EGFP (but not Mu-EGFP) is transiently expressed prior to or shortly after infection with Ad2 did not express late adenovirus genes. Further investigation suggested this might be due to a failure to express pre-terminal protein (preTP) from the E2 region, despite expression of another E2 protein, DBP. Deletion mutagenesis identified a highly conserved region in the C terminus of preMu responsible for these observations. Thus our data suggest that preMu may play a role in modulating accumulation of proteins from the E2 region.

Adenovirus E2 Proteins↗
PubMed2003

Nuclear perturbations following adenovirus infection.

Adenoviruses are processed and assembled in the nuclei of infected cells and thereby produce significant perturbations to their structure and function. As the complex interactions that occur in the nuclei of uninfected cells are not yet fully understood many of the changes seen on infection have been described mainly in morphological terms. This chapter attempts to place more recent findings into this context and demonstrates that adenoviruses are able to hijack many cellular processes and enzymes to their advantage. In particular, modifications to nuclear PODs and nucleoli have more recently been explored in greater detail.

Adenoviridae Infections↗
PubMed2001

Design and synthesis of irreversible depsipeptidyl human rhinovirus 3C protease inhibitors.

Novel tripeptidyl C-terminal Michael acceptors with an ester replacement of the P(2)-P(3) amide bond were investigated as irreversible inhibitors of the human rhinovirus (HRV) 3C protease (3CP). When screened against HRV serotype-14 the best compound was shown to have very good 3CP inhibition (k(obs)/[I]=270,000M(-1)s(-1)) and potent in vitro antiviral activity (EC(50)=7.0nM).

3C Viral Proteases↗
PubMed2001

Mitochondrial protein p32 can accumulate in the nucleus.

Human p32 was first isolated associated with the splicing factor ASF/SF-2. The p32 protein is translated as pre-protein from which a mitochondrial import signal is cleaved off to create the mature p32. The majority of p32 is consequently found in the mitochondria. In this study we investigated extramitochondrial p32. An increased nuclear localisation of endogenous p32 was demonstrated as a response to leptomycin B or actinomycin D treatment of cells. Mature p32 gene and deletion mutants were cloned into enhanced green fluorescence protein reporter plasmids. On transfection, EGFP-p32 protein was mainly localised to the cytoplasm and to a lesser extent to the nucleus of transfected COS cells. Upon treatment with actinomycin D or leptomycin B, the EGFP-p32 protein accumulated in the nucleus. Deletion analysis indicated which regions of EGFP-p32 are involved in nuclear export and nuclear import.

Active Transport, Cell Nucleus↗
PubMed2001

Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core.

Promising advances in nonviral gene transfer have been made as a result of the production of cationic liposomes formulated with synthetic cationic lipids (cytofectins) that are able to transfect cells. However few cationic liposome systems have been examined for their ability to transfect CNS cells. Building upon our earlier use of cationic liposomes formulated from 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidyl-ethanolamine (DOPE), we describe studies using two cationic viral peptides, mu (mu) and Vp1, as potential enhancers for cationic liposome-mediated transfection. Mu is derived from the condensed core of the adenovirus and was selected to be a powerful nucleic acid charge neutralising and condensing agent. Vp1 derives from the polyomavirus and harbours a classical nuclear localisation signal (NLS). Vp1 proved disappointing but lipopolyplex mixtures formulated from pCMVbeta plasmid, mu peptide and DC-Chol/DOPE cationic liposomes were able to transfect an undifferentiated neuronal ND7 cell line with beta-galactosidase reporter gene five-fold more effectively than lipoplex mixtures prepared from pCMVbeta plasmid and DC-Chol/DOPE cationic liposomes. Mu was found to give an identical enhancement to cationic liposome-mediated transfection of ND7 cells as poly-L-lysine (pLL) or protamine sulfate (PA). The enhancing effects of mu were found to be even greater (six- to 10-fold) when differentiated ND7 cells were transfected with mu-containing lipopolyplex mixtures. Differentiated ND7 cells represent a simple ex vivo-like post-mitotic CNS cell system. Successful transfection of these cells bodes well for transfection of primary neurons and CNS cells in vivo. These findings have implications for experimental and therapeutic uses of cationic liposome-mediated delivery of nucleic acids to CNS cells.

Animals↗
PubMed2001

Adenovirus protein V induces redistribution of nucleolin and B23 from nucleolus to cytoplasm.

Adenovirus infection inhibits synthesis and processing of rRNA and redistributes nucleolar antigens. Adenovirus protein V associates with nucleoli in infected cells. This study delineates regions of protein V independently capable of nucleolar targeting. Also, evidence is presented that protein V has the unique property of relocating nucleolin and B23 to the cytoplasm when transiently expressed on its own in uninfected cells. Point mutation analysis indicates a role for the C terminus of protein V in the redirection of nucleolin and B23 to the cytoplasm. This is the first time an adenovirus protein has been shown to have a direct effect on nucleolar antigens in isolation from viral infection. Moreover, adenovirus protein V is the first protein demonstrated to be capable of redirecting nucleolin and B23 to the cytoplasm.

Adenovirus Infections, Human↗
PubMed2000

Substituted benzamide inhibitors of human rhinovirus 3C protease: structure-based design, synthesis, and biological evaluation.

A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate. alpha,beta-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC(50) 0.60 microM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

3C Viral Proteases↗
PubMed2000

Prayer and spirituality.

Many patients with arthritis are strongly influenced by religious beliefs and often participate in religious healing activities such as prayer and worship attendance. Scientific studies demonstrate, and most patients confirm, that faith and involvement in religious healing activities can be helpful in preventing and treating illness, recovering from surgery, reducing pain, and improving quality of life. To improve the care of patients, clinicians should develop a patient-centered, spiritually sensitive form of medical practice in which religious issues are addressed gently and appropriately with dignity, respect, and integrity.

Activities of Daily Living↗
PubMed2000

Interaction between herpes simplex virus type 1 IE63 protein and cellular protein p32.

The herpes simplex virus type 1 (HSV-1) immediate-early gene IE63 (ICP27), the only HSV-1 regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far, is a multifunctional protein which regulates transcriptional and posttranscriptional processes. One of its posttranscriptional effects is the inhibition of splicing of viral and cellular transcripts. We previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and casein kinase 2 (CK2) as two protein partners of IE63 (H. Bryant et al., J. Biol. Chem. 274:28991-28998, 1999). Here, using a yeast two-hybrid assay, we identify another partner of IE63, the cellular protein p32. Confirmation of this interaction was provided by coimmunoprecipitation from virus-infected cells and recombinant p32 binding assays. A p32-hnRNP K-CK2 complex, which required IE63 to form, was isolated from HSV-1-infected cells, and coimmunoprecipitating p32 was phosphorylated by CK2. Expression of IE63 altered the cytoplasmic distribution of p32, with some now colocalizing with IE63 in the nuclei of infected and transfected cells. As p32 copurifies with splicing factors and can inhibit splicing, we propose that IE63 together with p32, possibly with other IE63 partner proteins, acts to disrupt or regulate pre-mRNA splicing. As well as contributing to host cell shutoff, this effect could facilitate splicing-independent nuclear export of viral transcripts.

Amino Acid Sequence↗
PubMed2000

Effects of intercessory prayer on patients with rheumatoid arthritis.

BACKGROUND: Many individuals pray during times of illness, but the clinical effects of prayer are not well-understood. METHODS: We prospectively studied a cohort of 40 patients (mean age, 62 years; 100% white; 82% women) at a private rheumatology practice. All had class II or III rheumatoid arthritis and took stable doses of antirheumatic medications. All received a 3-day intervention, including 6 hours of education and 6 hours of direct-contact intercessory prayer. Nineteen randomly selected sample patients had 6 months of daily, supplemental intercessory prayer by individuals located elsewhere. Ten arthritis-specific outcome variables were measured at baseline and at 3-month intervals for 1 year. RESULTS: Patients receiving in-person intercessory prayer showed significant overall improvement during 1-year follow-up. No additional effects from supplemental, distant intercessory prayer were found. CONCLUSIONS: In-person intercessory prayer may be a useful adjunct to standard medical care for certain patients with rheumatoid arthritis. Supplemental, distant intercessory prayer offers no additional benefits.

Adult↗
PubMed1999

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes.

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having alpha,beta-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

3C Viral Proteases↗
PubMed1999

Structure-based design of irreversible, tripeptidyl human rhinovirus 3C protease inhibitors containing N-methyl amino acids.

Tripeptide-derived molecules incorporating N-methyl amino acid residues and C-terminal Michael acceptor moieties were evaluated as irreversible inhibitors of the cysteine-containing human rhinovirus 3C protease (3CP). Such compounds displayed good 3CP inhibition activity (k(obs)/[I] up to 610,000 M(-1) s(-1)) and potent in vitro antiviral properties (EC50 approaching 0.03 microM) when tested against HRV serotype-14.

3C Viral Proteases↗
PubMed1999

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 3. Structure-activity studies of ketomethylene-containing peptidomimetics.

The structure-based design, chemical synthesis, and biological evaluation of various ketomethylene-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The ketomethylene-containing inhibitors typically display slightly reduced 3CP inhibition activity relative to the corresponding peptide-derived molecules, but they also exhibit significantly improved antiviral properties. Optimization of the ketomethylene-containing compounds is shown to provide several highly active 3C protease inhibitors which function as potent antirhinoviral agents (EC90 = <1 microM) against multiple virus serotypes in cell culture.

3C Viral Proteases↗
PubMed1999

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements.

The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC90 approximately 0.10 microM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

3C Viral Proteases↗
PubMed1999

The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation.

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.

Carrier Proteins↗