Discovery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases

A series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases is described. The compounds demonstrated potency with IC₅₀ values in the low nanomolar range in vitro while the lead compound also showed in vivo activity against various human tumor xenograft models in mice. Further exploration of this class of compounds is underway.     

The Rous sarcoma virus Env glycoprotein contains a highly conserved motif homologous to tyrosine-based endocytosis signals and displays an unusual internalization phenotype

The cytoplasmic domains of retroviral transmembrane (TM) glycoproteins contain conserved sequence motifs that resemble tyrosine-based (YXXO-type) endocytosis signals. We have previously described a mutant Rous sarcoma virus (RSV) Env protein, Env-mu26, with an L165R mutation in the membrane-spanning domain (MSD) of TM, that exhibited dramatically decreased steady-state surface expression (G. L. Davis and E. Hunter, J. Cell Biol. 105:1191-1203, 1987; P. B. Johnston, J. Y. Dong, and E. Hunter, Virology 206:353-361, 1995). We now demonstrate that the tyrosine of the Y(190)RKM motif in the RSV TM cytoplasmic domain is crucial for the mu26 phenotype and is part of an efficient internalization signal in the context of a mutant MSD. In contrast, despite the presence of the Y(190)RKM motif, wild-type RSV Env is constitutively internalized at a slow rate (1.1%/min) more characteristic of bulk uptake during membrane turnover than of active clustering into endocytic vesicles. The mu26 mutation and two MSD mutations that abrogate palmitoylation of TM resulted in enhanced Env endocytosis indicative of active concentration into coated pits. Surprisingly, an Env-Y190A mutant was apparently excluded from coated pits since its uptake rate of 0.3%/min was significantly below that expected for the bulk rate. We suggest that in RSV Env an inherently functional endocytosis motif is silenced by a counteracting determinant in the MSD that acts to prevent clustering of Env into endocytic vesicles. Mutations in either the cytoplasmic tail or the MSD that inactivate one of the two counteracting signals would thus render the remaining determinant dominant.     

Association of Moraxella ovis with keratoconjunctivitis in mule deer and moose in Wyoming

Six cases of infectious keratoconjunctivitis (IKC) in mule deer (Odocoileus hemionus) and moose (Alces alces) in Wyoming (USA) were investigated during fall and winter of 1995 and 1996. Excessive lacrimation, mucopurulent conjunctivitis, keratitis, and corneal opacity were observed in mule deer. Moose had severe mucopurulent conjunctivitis, keratitis, and corneal ulceration. Hemolytic, non-piliated Moraxella ovis was isolated from two mule deer and two moose. We attempted to reproduce IKC in three mule deer fawns using an isolate of M. ovis from a clinically affected mule deer. These fawns did not develop clinical signs of infection and the bacterium was not reisolated from inoculated deer. Inoculated deer may not have developed clinical signs because deer were not exposed to ultraviolet light or mechanical insult before inoculation. In addition, the isolate used for inoculation may have lost virulence factors through passage, or M. ovis may not have been the primary pathogen responsible for clinical disease in the natural cases of IKC we investigated. The etiology of IKC in free-ranging wild ruminants remains poorly understood.     

Strongyloides robustus and the northern sympatric populations of northern (Glaucomys sabrinus) and southern (G. volans) flying squirrels

Within North America, northern (Glaucomys sabrinus) and southern (Glaucomys volans) flying squirrels occupy distinct ranges with limited overlap. Sympatry in northern latitudes coincides with northern hardwood vegetation from Minnesota to New England. Strongyloides robustus is an intestinal parasite that infects both species but appears to be deleterious only to northern flying squirrels. As a result, S. robustus could be a critical determinant of flying squirrel population characteristics in at least some areas of sympatry. However, cold weather could potentially limit the distribution of S. robustus in northern climates. Therefore, we assessed fecal samples from both flying squirrel species to determine the presence of the nematode in Wisconsin. Strongyloides robustus was found in 12 flying squirrel scat samples and infected 52% of southern flying squirrels and 11% of northern flying squirrels. Prevalence of S. robustus infection for northern flying squirrels was substantially lower than previously reported from more southern regions. This is the northernmost documentation of S. robustus in flying squirrels and the first documentation of S. robustus parasitizing flying squirrels in Wisconsin.     

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.     

Prediction of "aggregation-prone" and "aggregation-susceptible" regions in proteins associated with neurodegenerative diseases

Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.     

Analysis of the cleavage site of the human immunodeficiency virus type 1 glycoprotein: requirement of precursor cleavage for glycoprotein incorporation.

Endoproteolytic cleavage of the glycoprotein precursor to the mature SU and TM proteins is an essential step in the maturation of retroviral glycoproteins. Cleavage of the precursor polyprotein occurs at a conserved, basic tetrapeptide sequence and is carried out by a cellular protease. The glycoprotein of the human immunodeficiency virus type 1 contains two potential cleavage sequences immediately preceding the N terminus of the TM protein. To determine the functional significance of these two potential cleavage sites, a series of mutations has been constructed in each site individually, as well as in combinations that altered both sites simultaneously. A majority of the mutations in either potential cleavage site continued to allow efficient cleavage when present alone but abrogated cleavage of the precursor when combined. Despite being transported efficiently to the cell surface, these cleavage-defective glycoproteins were unable to initiate cell-cell fusion and viruses containing them were not infectious. Viruses that contained glycoproteins with a single mutation, and that retained the ability to be processed, were capable of mediating a productive infection, although infectivity was impaired in several of these mutants. Protein analyses indicated that uncleaved glycoprotein precursors were inefficiently incorporated into virions, suggesting that cleavage of the glycoprotein may be a prerequisite to incorporation into virions. The substitution of a glutamic acid residue for a highly conserved lysine residue in the primary cleavage site (residue 510) had no effect on glycoprotein cleavage or function, even though it removed the only dibasic amino acid pair in this site. Peptide sequencing of the N terminus of gp41 produced from this mutant glycoprotein demonstrated that cleavage continued to take place at this site. These results, demonstrating that normal cleavage of the human immunodeficiency virus type 1 glycoprotein can occur when no dibasic sequence is present at the cleavage site, raise questions about the specificity of the cellular protease that mediates this cleavage and suggest that cleavage of the glycoprotein is required for efficient incorporation of the glycoprotein into virions.     

Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein define a requirement for dibasic residues for intracellular cleavage.

We investigated the amino acid sequence requirements for intracellular cleavage of the Rous sarcoma virus glycoprotein precursor by introducing mutations into the region encoding the cleavage recognition site (Arg-Arg-Lys-Arg). In addition to mutants G1 (Arg-Arg-Glu-Arg) and Dr1 (deletion of all four codons) that we have reported on previously (L. G. Perez and E. Hunter, J. Virol. 61:1609-1614, 1987), we constructed two additional mutants, AR1 (Arg-Arg-Arg-Arg), in which the highly conserved lysine is replaced by an arginine, and S19 (Ser-Arg-Glu-Arg), in which no dibasic pairs remain. The results of these studies demonstrate that when the cleavage sequence is deleted (Dr1) or modified to contain unpaired basic residues (S19), intracellular cleavage of the glycoprotein precursor is completely blocked. This demonstrates that the cellular endopeptidase responsible for cleavage has a stringent requirement for the presence of a pair of basic residues (Arg-Arg or Lys-Arg). Furthermore, it implies that the cleavage enzyme is not trypsinlike, since it is unable to recognize arginine residues that are sensitive to trypsin action. Substitution of the mutated genes into a replication-competent avian retrovirus genome showed that cleavage of the glycoprotein precursor was not required for incorporation into virions but was necessary for infectivity. Treatment of BH-RCAN-S19-transfected turkey cells with low levels of trypsin resulted in the release of infectious virus, demonstrating that exogenous cleavage could generate a biologically active glycoprotein molecule.     

Truncation of the human immunodeficiency virus type 1 transmembrane glycoprotein cytoplasmic domain blocks virus infectivity.

Human immunodeficiency virus type 1 contains a transmembrane glycoprotein with an unusually long cytoplasmic domain. To determine the role of this domain in virus replication, a series of single nucleotide changes that result in the insertion of premature termination codons throughout the cytoplasmic domain has been constructed. These mutations delete from 6 to 192 amino acids from the carboxy terminus of gp41 and do not affect the amino acid sequence of the regulatory proteins encoded by rev and tat. The effects of these mutations on glycoprotein biosynthesis and function as well as on virus infectivity have been examined in the context of a glycoprotein expression vector and the viral genome. All of the mutant glycoproteins were synthesized, processed, and transported to the cell surface in a manner similar to that of the wild-type glycoprotein. With the exception of mutants that remove the membrane anchor domain, all of the mutant glycoproteins retained the ability to cause fusion of CD4-bearing cells. However, deletion of more than 19 amino acids from the C terminus of gp41 blocked the ability of mutant virions to infect cells. This defect in virus infectivity appeared to be due at least in part to a failure of the virus to efficiently incorporate the truncated glycoprotein. Similar data were obtained for mutations in two different env genes and two different target cell lines. These results indicate that the cytoplasmic domain of gp41 plays a critical role during virus assembly and entry in the life cycle of human immunodeficiency virus type 1.     

Rat Gene Mapping Using Pcr-Analyzed Microsatellites

One hundred and seventy-four rat loci which contain short tandem repeat sequences were extracted from the GenBank or EMBL data bases and used to define primers for amplification by the polymerase chain reaction (PCR) of the microsatellite regions, creating PCR-formatted sequence-tagged microsatellite sites (STMSs). One hundred and thirty-four STMSs for 118 loci, including 6 randomly cloned STMSs, were characterized: (i) PCR-analyzed loci were assigned to specific chromosomes using a panel of rat x mouse somatic cell hybrid clones. (ii) Length variation of the STMSs among 8 inbred rat strains could be visualized at 85 of 107 loci examined (79.4%). (iii) A genetic map, integrating biochemical, coat color, mutant and restriction fragment length polymorphism loci, was constructed based on the segregation of 125 polymorphic markers in seven rat backcrosses and in two F2 crosses. Twenty four linkage groups were identified, all of which were assigned to a defined chromosome. As a reflection of the bias for coding sequences in the public data bases, the STMSs described herein are often associated with genes. Hence, the genetic map we report coincides with a gene map. The corresponding map locations of the homologous mouse and human genes are also listed for comparative mapping purposes.