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Fred E Cohen

Publications and source records attributed to Fred E Cohen.

At least 19 recordsLinked to original sources
PubMed2007

Structure-activity relationship study of prion inhibition by 2-aminopyridine-3,5-dicarbonitrile-based compounds: parallel synthesis, bioactivity, and in vitro pharmacokinetics.

2-Aminopyridine-3,5-dicarbonitrile compounds were previously identified as mimetics of dominant-negative prion protein mutants and inhibit prion replication in cultured cells. Here, we report findings from a comprehensive structure-activity relationship study of the 6-aminopyridine-3,5-dicarbonitrile scaffold. We identify compounds with significantly improved bioactivity (approximately 40-fold) against replication of the infectious prion isoform (PrPSc) and suitable pharmacokinetic profiles to warrant evaluation in animal models of prion disease.

Aminopyridines↗
PubMed2006

Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes.

On passaging synthetic prions, two isolates emerged with incubation times differing by nearly 100 days. Using conformational-stability assays, we determined the guanidine hydrochloride (Gdn.HCl) concentration required to denature 50% of disease-causing prion protein (PrP(Sc)) molecules, denoted as the [Gdn.HCl](1/2) value. For the two prion isolates enciphering shorter and longer incubation times, [Gdn.HCl](1/2) values of 2.9 and 3.7 M, respectively, were found. Intrigued by this result, we measured the conformational stabilities of 30 prion isolates from synthetic and naturally occurring sources that had been passaged in mice. When the incubation times were plotted as a function of the [Gdn.HCl](1/2) values, a linear relationship was found with a correlation coefficient of 0.93. These findings demonstrate that (i) less stable prions replicate more rapidly than do stable prions, and (ii) a continuum of PrP(Sc) structural states enciphers a multitude of incubation-time phenotypes. Our data argue that cellular machinery must exist for propagating a large number of different PrP(Sc) conformers, each of which enciphers a distinct biological phenotype as reflected by a specific incubation time. The biophysical explanation for the unprecedented plasticity of PrP(Sc) remains to be determined.

Animals↗
PubMed2006

Structure-activity relationship study of 9-aminoacridine compounds in scrapie-infected neuroblastoma cells.

A focused library of variously substituted 9-aminoacridine compounds was screened for bioactivity against accumulation of the infectious prion protein isoform, denoted PrP(Sc), in a cell model of prion replication. The efficacy of compounds against PrP(Sc) accumulation was influenced by both substituents of the distal tertiary amine and acridine heterocycle, while cellular cytotoxicity was encoded in the acridine heterocycle substituents.

Aminacrine↗
PubMed2006

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.

Amino Acid Sequence↗
PubMed2006

Developing therapeutics for the diseases of protein misfolding.

Our current structural and biologic understanding of the misfolding diseases has restricted the development of therapies that target these diseases at a molecular level. The prion diseases are illustrative of this group of misfolding disorders and provide a model system for therapeutic intervention. Strategies to inhibit the replication and accumulation of the prion protein are being developed and have entered animal and clinical studies. Due to the underlying molecular basis of this disease class, many of the therapeutic approaches used to target prion misfolding have parallels in other misfolding diseases.

Alzheimer Disease↗
PubMed2006

Searching for new antimalarial therapeutics amongst known drugs.

The need to discover and develop new antimalarial therapeutics is overwhelming. The annual mortality attributed to malaria, currently approximately 2.5 million, is increasing due primarily to widespread resistance to currently used drugs. One strategy to identify new treatment alternatives for malaria is to examine libraries of diverse compounds for the possible identification of novel scaffolds. Beginning with libraries of drug or drug-like compounds is an ideal starting point because, in the case of approved drugs, substantial pharmacokinetic and toxicologic data should be available for each compound series. We have employed a high-throughput screen of the MicroSource Spectrum and Killer Collections, a library of known drugs, bioactive compounds, and natural products. Our screening assay identifies compounds that inhibit growth of Plasmodium falciparum cultured in human erythrocytes. We have identified 36 novel inhibitors of P. falciparum, of which 19 are therapeutics, and five of these drugs exhibit effective 50% inhibitory concentrations within similar ranges to therapeutic serum concentrations for their recently indicated uses: propafenone, thioridazine, chlorprothixene, perhexiline, and azlocillin. The findings we report here indicate that this is an effective strategy to identify novel scaffolds and therefore aid in antimalarial drug discovery efforts.

Animals↗
PubMed2005

Apolipoprotein (apo) E4 enhances amyloid beta peptide production in cultured neuronal cells: apoE structure as a potential therapeutic target.

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoform-specific effects on the deposition or clearance of amyloid beta (Abeta) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on Abeta production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased Abeta production in B103-APP cells to a greater extent than lipid-poor apoE3 (60% vs. 30%) due to more pronounced stimulation of APP recycling by apoE4 than apoE3. The difference in Abeta production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptor-related protein (LRP) pathway, or by reducing LRP expression by small interference RNA. The differences were also attenuated by replacing Arg-61 with threonine in apoE4 or pretreating apoE4 with small molecules, both of which abolish apoE4 intramolecular domain interaction. Thus, apoE4 appears to modulate APP processing and Abeta production through both the LRP pathway and domain interaction. These findings provide insights into why apoE4 is associated with increased risk for Alzheimer's disease and may represent a potential target for drug development.

Amyloid beta-Peptides↗
PubMed2005

Strain-specified characteristics of mouse synthetic prions.

Synthetic prions were produced in our laboratory by using recombinant mouse prion protein (MoPrP) composed of residues 89-230. The first mouse synthetic prion strain (MoSP1) was inoculated into transgenic (Tg) 9949 mice expressing N-terminally truncated MoPrP(Delta23-88) and WT FVB mice expressing full-length MoPrP. On first and second passage in Tg9949 mice, MoSP1 prions caused disease in 516 +/- 27 and 258 +/- 25 days, respectively; numerous, large vacuoles were found in the brainstem and gray matter of the cerebellum. MoSP1 prions passaged in Tg9949 mice were inoculated into FVB mice; on first and second passage, the FVB mice exhibited incubation times of 154 +/- 4 and 130 +/- 3 days, respectively. In FVB mice, vacuolation was less intense but more widely distributed, with numerous lesions in the hippocampus and cerebellar white matter. This constellation of widespread neuropatho-logic changes was similar to that found in FVB mice inoculated with Rocky Mountain Laboratory (RML) prions, a strain derived from a sheep with scrapie. Conformational stability studies showed that the half-maximal GdnHCl (Gdn1/2) concentration for denaturation of MoSP1 prions passaged in Tg9949 mice was approximately 4.2 M; passage in FVB mice reduced the Gdn1/2 value to approximately 1.7 M. RML prions passaged in either Tg9949 or FVB mice exhibited Gdn1/2 values of approximately 1.8 M. The incubation times, neuropathological lesion profiles, and Gdn1/2 values indicate that MoSP1 prions differ from RML and many other prion strains derived from sheep with scrapie and cattle with bovine spongiform encephalopathy.

Animals↗
PubMed2004

Synthetic mammalian prions.

Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of beta sheet-rich structures. Fibrils consisting of recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.

Amyloid↗
PubMed2004

Evidence for assembly of prions with left-handed beta-helices into trimers.

Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt a cross-beta structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform (PrP(Sc)) of the prion protein, designated PrP 27-30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right- or left-handed beta-helical models. From a study of 119 all-beta folds observed in globular proteins, we have now determined that, if PrP(Sc) follows a known protein fold, it adopts either a beta-sandwich or parallel beta-helical architecture. With increasing evidence arguing for a parallel beta-sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed beta-helical fold. Left-handed beta-helices readily form trimers, providing a natural template for a trimeric model of PrP(Sc). This trimeric model accommodates the PrP sequence from residues 89-175 in a beta-helical conformation with the C terminus (residues 176-227), retaining the disulfide-linked alpha-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27-30 crystals, positioning residues 141-176 and the N-linked sugars appropriately. Our parallel left-handed beta-helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed beta-helical model for PrP(Sc) may provide important clues to the structure of filaments found in some other neurodegenerative diseases.

Amino Acid Sequence↗
PubMed2004

Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.

Because of their sensitivity to solubilizing detergents, membrane protein assemblies are difficult to study. We describe a protocol that covalently conserves protein interactions through time-controlled transcardiac perfusion cross-linking (tcTPC) before disruption of tissue integrity. To validate tcTPC for identifying protein-protein interactions, we established that tcTPC allowed stringent immunoaffinity purification of the gamma-secretase complex in high salt concentrations and detergents and was compatible with mass spectrometric identification of cross-linked aph-1, presenilin-1 and nicastrin. We then applied tcTPC to identify more than 20 proteins residing in the vicinity of the cellular prion protein (PrPC), suggesting that PrP is embedded in specialized membrane regions with a subset of molecules that, like PrP, use a glycosylphosphatidylinositol anchor for membrane attachment. Many of these proteins have been implicated in cell adhesion/neuritic outgrowth, and harbor immunoglobulin C2 and fibronectin type III-like motifs.

Amino Acid Sequence↗
PubMed2004

Automated extraction of mutation data from the literature: application of MuteXt to G protein-coupled receptors and nuclear hormone receptors.

MOTIVATION: The amount of genomic and proteomic data that is published daily in the scientific literature is outstripping the ability of experimental scientists to stay current. Reviews, the traditional medium for collating published observations, are also unable to keep pace. For some specific classes of information (e.g. sequences and protein structures), obligatory data deposition policies have helped. However, a great deal of other valuable information is spread throughout the literature hindering coherent access. We are involved in the Molecular Class-Specific Information System (MCSIS) project, a collaborative effort to design and automate the maintenance of protein family databases. The first two databases, the GPCRDB and NucleaRDB, are focused on G protein-coupled receptors (GPCRs) and nuclear hormone receptors (NRs), respectively. The main aim of the MCSIS project is to gather heterogeneous data from across a variety of electronic and literature sources in order to draw new inferences about the target protein families. RESULTS: We present a computational method that identifies and extracts mutation data from the scientific literature. We focused on the extraction of single point mutations for the GPCR and NR superfamilies. After validation by plausibility filters, the mutation data is integrated into the corresponding MCSIS where it is combined with structural and sequence information already stored in these databases. We extracted and validated 2736 true point mutations from 914 articles on GPCRs and 785 true point mutations from 1094 articles on NRs. The current version of our automated extraction algorithm identifies 49.3% of the GPCR point mutations with a specificity of 87.9%, and 64.5% of the NR point mutations with a specificity of 85.8%. MuteXt routinely analyzes 100 electronic articles in approximately 1 h.

Algorithms↗
PubMed2004

Mutant PrPSc conformers induced by a synthetic peptide and several prion strains.

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP(Sc)(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4 degrees C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP(Sc)(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a beta-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP(Sc)(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP(Sc) in the brains of Tg196 mice. Our results argue that MoPrP(Sc)(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP(C)(P101L) as well as in Tg196 mice expressing low levels of MoPrP(C)(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a beta-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.

Animals↗
PubMed2003

Therapeutic approaches to protein-misfolding diseases.

Several sporadic and genetic diseases are caused by protein misfolding. These include cystic fibrosis and other devastating diseases of childhood as well as Alzheimer's, Parkinson's and other debilitating maladies of the elderly. A unified view of the molecular and cellular pathogenesis of these conditions has led to the search for chemical chaperones that can slow, arrest or revert disease progression. Molecules are now emerging that link our biophysical insights with our therapeutic aspirations.

Alzheimer Disease↗
PubMed2003

New local potential useful for genome annotation and 3D modeling.

A new potential energy function representing the conformational preferences of sequentially local regions of a protein backbone is presented. This potential is derived from secondary structure probabilities such as those produced by neural network-based prediction methods. The potential is applied to the problem of remote homolog identification, in combination with a distance-dependent inter-residue potential and position-based scoring matrices. This fold recognition jury is implemented in a Java application called JThread. These methods are benchmarked on several test sets, including one released entirely after development and parameterization of JThread. In benchmark tests to identify known folds structurally similar to (but not identical with) the native structure of a sequence, JThread performs significantly better than PSI-BLAST, with 10% more structures identified correctly as the most likely structural match in a fold library, and 20% more structures correctly narrowed down to a set of five possible candidates. JThread also improves the average sequence alignment accuracy significantly, from 53% to 62% of residues aligned correctly. Reliable fold assignments and alignments are identified, making the method useful for genome annotation. JThread is applied to predicted open reading frames (ORFs) from the genomes of Mycoplasma genitalium and Drosophila melanogaster, identifying 20 new structural annotations in the former and 801 in the latter.

Animals↗
PubMed2003

Potent inhibition of scrapie prion replication in cultured cells by bis-acridines.

Prion diseases are characterized by an accumulation of PrP(Sc), a misfolded isoform of the normal cellular prion protein, PrP(C). We previously reported the bioactivity of acridine-based compounds against PrP(Sc) replication in scrapie-infected neuroblastoma cells and now report the improved potency of bis-acridine compounds. Bis-acridines are characterized by a dimeric motif, comprising two acridine heterocycles tethered by a linker. A library of bis-(6-chloro-2-methoxy-acridin-9-yl) and bis-(7-chloro-2-methoxy-benzo[b][1,5]naphthyridin-10-yl) analogs was synthesized to explore the effect of structurally diverse linkers on PrP(Sc) replication in scrapie-infected neuroblastoma cells. Structure-activity analysis revealed that linker length and structure are important determinants for inhibition of prion replication in cultured scrapied cells. Three bis-acridine analogs, (6-chloro-2-methoxy-acridin-9-yl)-(3-[4-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-piperazin-1-yl]-propyl)-amine, N,N'-bis-(6-chloro-2-methoxy-acridin-9-yl)-1,8-diamino-3,6-dioxaoctane, and (1-[[4-(6-chloro-2-methoxy-acridin-9-ylamino)-butyl]-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-carbamoyl]-ethyl)-carbamic acid tert-butyl ester, showed half-maximal inhibition of PrP(Sc) formation at 40, 25, and 30 nM, respectively, and were not cytotoxic to uninfected neuroblastoma cells at concentrations of 500 nM. Our data suggest that bis-acridine analogs may provide a potent alternative to the acridine-based compound quinacrine, which is currently under clinical evaluation for the treatment of prion disease.

Acridines↗
PubMed2003

Expression profiling of the schizont and trophozoite stages of Plasmodium falciparum with a long-oligonucleotide microarray.

BACKGROUND: The worldwide persistence of drug-resistant Plasmodium falciparum, the most lethal variety of human malaria, is a global health concern. The P. falciparum sequencing project has brought new opportunities for identifying molecular targets for antimalarial drug and vaccine development. RESULTS: We developed a software package, ArrayOligoSelector, to design an open reading frame (ORF)-specific DNA microarray using the publicly available P. falciparum genome sequence. Each gene was represented by one or more long 70 mer oligonucleotides selected on the basis of uniqueness within the genome, exclusion of low-complexity sequence, balanced base composition and proximity to the 3' end. A first-generation microarray representing approximately 6,000 ORFs of the P. falciparum genome was constructed. Array performance was evaluated through the use of control oligonucleotide sets with increasing levels of introduced mutations, as well as traditional northern blotting. Using this array, we extensively characterized the gene-expression profile of the intraerythrocytic trophozoite and schizont stages of P. falciparum. The results revealed extensive transcriptional regulation of genes specialized for processes specific to these two stages. CONCLUSIONS: DNA microarrays based on long oligonucleotides are powerful tools for the functional annotation and exploration of the P. falciparum genome. Expression profiling of trophozoites and schizonts revealed genes associated with stage-specific processes and may serve as the basis for future drug targets and vaccine development.

Animals↗