Barbara A. Cottrell

Substrate-targeting γ-secretase modulators

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer’s disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β deposition in Alzheimer’s disease. These data also demonstrate the existence and feasibility of ‘substrate targeting’ by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of ‘druggable’ targets.

The amino acid sequence of the α-chain of human fibrinogen

The amino acid sequence of the human fibrinogen α-chain reveals a structure that can be divided into three zones of unique amino acid composition. The middle of these contains the two primary α-chain cross-linking acceptor sites and consists of a remarkable series of internal duplications.

Safety, tolerability, pharmacokinetics, and Aβ levels after short-term administration of R-flurbiprofen in healthy elderly individuals

To evaluate the safety and tolerability and pharmacokinetic properties of R-flurbiprofen (Tarenflurbil) in normal elderly individuals and to determine the effect of the drug on amyloid beta 42 (Aβ42) levels, we conducted a double-blind, placebo-controlled study of 48 healthy subjects aged 55 to 80. Three successive cohorts were randomized to doses of 400, 800, or 1600 mg/d, or placebo, given as 2 divided doses for 21 days. Blood and cerebrospinal fluid were collected for pharmacokinetic studies and measurement of Aβ levels at baseline and on day 21. R-flurbiprofen was well-tolerated at all 3 doses. The compound penetrated the blood-brain barrier in a dose-dependent manner. From baseline to 21 days, comparisons between study groups revealed no significant differences in changes of cerebrospinal fluid Aβ42 levels and no significant differences in changes of plasma Aβ42 levels at the time of trough drug level at 21 days of treatment. Further analysis of drug concentration-response for plasma samples showed that at the time of peak plasma concentration, higher plasma drug concentration was related to lower Aβ42 plasma levels (P=0.016). R-flurbiprofen had an excellent safety profile and showed dose-dependent central nervous system penetration. Exploratory analyses of plasma Aβ and peak drug levels suggested a short-term effect in plasma that warrants independent verification. The safety, tolerability, and pharmacokinetic profile of R-flurbiprofen in these older individuals support the ongoing studies of this compound in patients with Alzheimer disease.

SYNTHETIC PEPTIDES MODELED ON FIBRIN POLYMERIZATION SITES

The transformation of fibrinogen into a fibrin clot is initiated by the proteolytic enzyme thrombin that cleaves fibrinopeptides A and B from the amino termini of the a and B chains of fibrinogen.’ The resulting fibrin monomers spontaneously polymerize to form a noncovalently bound gel stabilized by intermolecular cross-linking in the presence of activated factor XI11 and calcium. Thirty years ago Ferry * proposed that polymerization proceeds through two associative processes involving, initially, an end-toend interaction leading to the formation of intermediate polymers, and then, a lateral aggregation of these intermediate strands. He postulated that intermediate polymers two molecules thick arranged in a staggered overlapping fashion might form as a result of interactions involving the positive amino termini that are exposed by thrombin. It was later found that all six amino termini of fibrinogen are located in a single disulfide-bonded cyanogen bromide fragment, the “amino-terminal disulfide knot” (NDSK) ,3 which was found to be immunologically cross-reactive with the plasmin-derived fragment E.4 Recent electron microscopic studies utilizing antibodies that are directed toward various domains of fibrinogen have supported previous notions that this amino terminal domain comprises the central domain 5-1 of the triglobular fibrinogen molecule first observed by Hall and Slayter,8 and more recently by others.s-ll A decade ago it was proposed that release of fibrinopeptides from the central domain of a triglobular molecule allows reciprocal interactions of the central domains with the terminal domains of neighboring m01ecules.~~ This notion was subsequently verified by experiments in which fibrinogen and fragment D, the plasmin-derived fragment that approximates the terminal domain, bound to both fibrin monomer and thrombin activated NDSK immobilized on Sepharose.12 Amino acid sequence studies have revealed that the amino terminal regions of the a and , 9 chains of fibrin that are exposed by fibrinopeptide release are highly conserved from species to specie^.^^-^^ In particular, the a chain of fibrin begins with the same tripeptide sequence, glycyl-L-prolyl-L-arginine (Gly-Pro-Arg), in all species studied, including the primitive lamprey. It is likely that important functional constraints related to fibrin polymerization have led to the evolutionary persistence of this moiety. Furthermore, the variant human fibrinogen Detroit, which exhibits defective polymerization, has a replacement of arginine by serine at the third residue from the amino terminus

The amino acid sequence of a 27-residue peptide feleased from the α-chain carboxy-terminus during the plasmic digestion of human fibrinogen

Abstract The amino acid sequence of a 27-residue peptide released during the early stages of the plasmin digestion of human fibrinogen has been determined. The corresponding cyanogen bromide fragment has also been isolated from the purified α-chains of fibrinogen, although a separable fraction of those chains lack the fragment, evidently because of in vivo degradation. The peptide is the carboxy-terminal segment of native α-chains.

Lysine 624 of the Amyloid Precursor Protein (APP) Is a Critical Determinant of Amyloid β Peptide Length SUPPORT FOR A SEQUENTIAL MODEL OF γ-SECRETASE INTRAMEMBRANE PROTEOLYSIS AND REGULATION BY THE AMYLOID β PRECURSOR PROTEIN (APP) JUXTAMEMBRANE REGION

Background: γ-Secretase modulators (GSMs) bind APP near lysine 624. Results: Mutation of lysine 624 shifts cleavage toward smaller Aβ with no effect on ϵ cleavage. Conclusion: The amino acid at 624 in substrates affects the final γ-secretase cut. Significance: γ-Secretase cleavage likely begins at ϵ and proceeds up the transmembrane until Aβ is released, and GSMs may modulate this process through lysine 624. γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimers disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ϵ, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1–42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1–40 to 1–33 without significant changes to ϵ cleavage. These results further support a model where ϵ cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Amino acid sequences of lamprey fibrinopeptides A and B and characterization of the junctions split by lamprey and mammalian thrombins

The amino acid sequences of the fibrinopeptides A and B from lamprey fibrinogen have been determined. The fibrinopeptide A is the shortest fibrinopeptide ever isolated, being comprised of only six amino acids. The fibrinopeptide B, on the other hand, is the largest fibrinopeptide characterized to date, having 36 amino acid residues and a cluster of covalently bound carbohydrate. As reported previously, lamprey fibrinogen is readily clotted by mammalian thrombins, but only the fibrinopeptide B is released during the process. Lamprey fibrinopeptide A is not released by mammalian thrombins and could only be removed with the use of lamprey thrombin. Firm proof that the lamprey fibrinopeptides A and B are the amino segments of the alpha and beta-chains respectively was obtained by a series of stepwise degradations on lamprey fibrinogen and lamprey fibrins produced in turn by the action of mammalian thrombin (fibrin B) and lamprey thrombin (fibrin A). These studies were supplemented by stepwise degradations on the individual Aalpha and Bbeta-chains. It the case of the lamprey Aalpha-chain it was also possible to release the 6-residue fibrinopeptide A from the isolated chain with lamprey thrombin and demonstrate that the newly exposed amino-terminal sequence begins with the Gly-Pro-Arg sequence characteristic of mammalian fibrin alpha-chains. In fact, the sequences on the fibrin side of both of the junctions split by thrombin(s) are highly conserved and virtually identical with those found in mammalian alpha and beta-chains.

Amino acid compositions of the subunit chains of lamprey fibrinogen. Evolutionary significance of some structural anomalies.

Our original objective in studying lamprey fibrinogen was embodied in the notion that the proteins of this ancient vertebrate might themselves by more primitive. As such, it was possible that the subunits of lamprey fibrinogen might have been more similar, one to another, than is the case in higher vertebrates, or even identical. Amino acid analysis of the individual polypeptide chains indicates, however, that the alpha, beta and gamma-chains are instead more dissimilar from each other than are the corresponding chains from human fibrinogen. This finding was somewhat surprising because regions of homology have been detected recently among those three chains when isolated from human fibrinogen, suggesting that all three chains have indeed descended from a common ancestor. The paradox is especially evidenced by the unusual amino acid composition of the lamprey alpha-chain, 45% of which is composed of glycine, serine and threonine. This unusual amino acid distribution may be involved in the anomalous behavior of these chains on sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The amino acid sequence of the carboxy-terminal 142 amino acids of the α-chain of human fibrinogen

Abstract The amino acid sequence of the carboxy-terminal 142 residues of the α-chain of human fibrinogen has been determined. The sequence was established by determining the structures of cyanogen bromide fragments and key overlap peptides isolated from enzymatic digests of α-chains. One of the cyanogen bromide fragments is a peptide we have previously shown to participate in the α-chain cross-linking system.

Lamprey fibrinopeptide B is A glycopeptide

Abstract One of the peptides released from lamprey fibrinogen during its transformation into fibrin has been found to contain covalently bound carbohydrate. The peptide, which also contains tyrosine O-sulfate, corresponds to the mammalian fibrinopeptide B and is the amino-terminal segment of the lamprey fibrinogen β-chain. As noted previously, this peptide is the only one released when lamprey fibrinogen is coltted by mammalian thrombin. Of the more than fifty sets of fibrinopeptides characterized from various species, this is the first one found to contain carbohydrate.