A presenilin-independent aspartyl protease prefers the gamma-42 site cleavage

beta-Amyloid peptides (Abeta40 and Abeta42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimer's disease (AD). The Abeta is derived from sequential cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. gamma-Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the gamma-site cleavage of APP. Secretion of both Abeta40 and Abeta42 is significantly reduced in PS1 knock-out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Abeta from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Abeta42 accumulation; thus the intracellular Abeta42 appears to be generated in a PS-independent manner. Here we present the first biochemical evidence indicating that Abeta, especially Abeta42, can be generated in the absence of PS based on an in vitrogamma-secretase assay employing membranes prepared from PS-deficient Blastocyst-derived (BD) cells. This PS-independent gamma-secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Abeta40 and Abeta42 generation, respectively. These results indicate that these PS-independent gamma-site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42-site cleavage over the 40-site cleavage, thereby generating Abeta42 as the predominant product.     

1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists: modifications of the arylpropylpiperidine side chains

The 4-(3-phenylprop-1-yl)piperidine moiety of the 1,3,4-trisubstituted pyrrolidine CCR5 antagonist 1 was modified with electron deficient aromatics as well as replacement of the benzylic methylene with sulfones, gem-difluoromethylenes and alcohols in an effort to balance the antiviral potency with reasonable pharmacokinetics.     

1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists. Part 4: synthesis of N-1 acidic functionality affording analogues with enhanced antiviral activity against HIV

A series of alpha-(pyrrolidin-1-yl)acetic acids is presented as selective and potent antivirals against HIV. Several of the pyrrolidine zwitterions demonstrated reasonable in vitro properties, enhanced antiviral activities and improved pharmacokinetic profiles over pyrrolidine 1.     

Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics.

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.     

Equivalent inhibition of half-site and full-site retroviral strand transfer reactions by structurally diverse compounds.

In vitro assay systems which use recombinant retroviral integrase (IN) and short DNA oligonucleotides fail to recapitulate the full-site integration reaction as it is known to occur in vivo. The relevance of using such circumscribed in vitro assays to define inhibitors of retroviral integration has not been formerly demonstrated. Therefore, we analyzed a series of structurally diverse inhibitors with respect to inhibition of both half-site and full-site strand transfer reactions with either recombinant or virion-produced IN. Half-site and full-site reactions catalyzed by avian myeloblastosis virus and human immunodeficiency virus type 1 (HIV-1) IN from virions are shown to be equivalently sensitive to inhibition by compounds which inhibit half-site reactions catalyzed by the recombinant HIV-1 IN. These studies therefore support the utility of using in vitro assays employing either recombinant or virion-derived IN to identify inhibitors of integration.     

Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

Characterization of the herpes simplex virus origin binding protein interaction with OriS.

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.