Patrick Harriott

A strategy for designing inhibitors of α-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders

Convergent biochemical and genetic evidence suggests that the formation of α‐synuclein (α‐syn) protein deposits is an important and, probably, seminal step in the development of Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been reported that transgenic animals overexpressing human α‐syn develop lesions similar to those found in the brain in PD, together with a progressive loss of dopaminergic cells and associated abnormalities of motor function. Inhibiting and/or reversing α‐syn self‐aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. We synthesized a library of overlapping 7‐mer peptides spanning the entire α‐syn sequence, and identified amino acid residues 64‒100 of α‐syn as the binding region responsible for its self‐ association. Modified short peptides containing α‐syn amino acid sequences from part of this binding region (residues 69‒72), named α‐syn inhibitors (ASI), were found to interact with full‐ length α‐syn and block its assembly into both early oligomers and mature amyloid‐like fibrils. We also developed a cell‐permeable inhibitor of α‐syn aggregation (ASID), using the polyarginine peptide delivery system. This ASID peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with α‐syn(A53T), a familial PD‐associated mutation. ASI peptides without this delivery system did not reverse levels of Fe(II)‐induced DNA damage. Furthermore, the ASID peptide increased (P<0.0005) the number of cells stained positive for Bcl‐2, while significantly (P<0.05) decreasing the percentage of cells stained positive for BAX. These short peptides could serve as lead compounds for the design of peptidomimetic drugs to treat PD and related disorders.

Glucagon-like peptide-1 analogues enhance synaptic plasticity in the brain: A link between diabetes and Alzheimer's disease

Type 2 diabetes has been identified as a risk factor for patients with Alzheimers disease. Insulin signalling is often impaired in Alzheimers disease, contributing to the neurodegenerative process. One potential strategy to help prevent this is the normalisation of insulin signalling in the brain. Therefore, the present study was designed to test the effects of novel enzyme-resistant analogues of the insulin-releasing incretin hormone, glucagon-like peptide 1 (GLP-1). The effects of Liraglutide (Victoza) and other novel GLP-1 analogues were tested on synaptic plasticity (LTP) in area CA1 of the hippocampus. At a dose of 15nmol in 5microl i.c.v., Liraglutide (P<0.005), Asp(7)GLP-1 (P<0.001), N-glyc-GLP-1 (P<0.01), and Pro(9)GLP-1 (P<0.001). In contrast, the GLP-1 receptor antagonist exendin(9-39)amide impaired LTP (P<0.001). Co-injection of exendin(9-39) and Liraglutide showed no effect on LTP. These results clearly demonstrate that Liraglutide and other GLP-1 analogues elicit effects on neurotransmission in the brain. Furthermore, GLP-1 peptides are not only effective in modulating insulin-release and achieving glycaemic control in type 2 diabetes, but are also effective in modulating synaptic plasticity. These findings are consistent with our previous observations that the novel analogue (Val(8))GLP-1 enhances LTP and reverses the impairments of LTP induced by beta-amyoid fragments. Therefore, the drug effects seen here could potentially ameliorate the impairments in neuronal communication and cognitive processes observed in Alzheimers disease.

A Sequence within the Cytoplasmic Tail of GpIIb Independently Activates Platelet Aggregation and Thromboxane Synthesis

All integrin α subunits contain a highly conserved KXGFFKR motif in their cytoplasmic domains that plays a crucial role in the regulation of integrin affinity for their ligands. We show that a lipid-modified peptide corresponding to the cytoplasmic region, 989–995, of the platelet integrin subunit glycoprotein GpIIb (αIIb), palmitoyl-KVGFFKR (Ppep; 10 μm), but not a similarly modified scrambled peptide (palmitoyl-FKFVRGK), can specifically induce platelet activation and aggregation equivalent to that of strong agonists such as thrombin. Ppep-induced aggregation is also associated with indices of platelet activation including thromboxane A2 (TXA2) synthesis (EC50 = 45 ± 5 μm), secretion of α-granules detected as enhanced surface expression of P-selectin (EC50 = 52 ± 8 μm), and conformational changes in GpIIb/IIIa measured by the monoclonal antibody, PAC-1 (EC50 = 3.7 ± 1 μm). The TXA2 receptor antagonist, SQ29548, PGE1, and the ADP scavenger, apyrase, differentially inhibit the aggregation response and TXA2 synthesis in response to Ppep. Similarly, GpIIb/IIIa antagonists (RO-449883 and integrelin), which inhibit aggregation by greater than 90%, have little effect on peptide-induced TXA2 synthesis, suggesting that this event is independent of fibrinogen binding to GpIIb/IIIa. Alanine-stepping of the Ppep sequence identifies GFFK(991–994) as the critical residues in all peptide-mediated events. We conclude that this peptide can imitate the cytoplasmic domain of GpIIb and initiate parallel but independent signaling pathways, one leading to ligand binding and platelet aggregation and the other to intracellular signaling events such as TXA2 synthesis and secretion.

Identification of potential activators of proteinase‐activated receptor‐21

In order to identify physiological activators of proteinase‐activated receptor‐2 (PAR‐2), a peptide chloromethane inhibitor (biotinyl‐Ser‐Lys‐Gly‐Arg‐CH2Cl) based on the cleavage site for activation of PAR‐2 was synthesised and tested with 12 trypsin‐like serine proteinases. The second‐order rate constant (k i/K i) for the formation of the covalent proteinase‐inhibitor complex varied by 2×105‐fold between the proteinases. Biotinyl‐Ser‐Lys‐Gly‐Arg‐CH2Cl reacted very rapidly with trypsin, acrosin from sperm and tryptase from mast cells: the k i/K i values with these proteinases were greater than 105 M−1 s−1. Thus, the specificity of these proteinases matched the sequence of the activation site of PAR‐2 and it can be concluded that these proteinases are potential physiological activators of PAR‐2.

Administration of an acylated GLP-1 and GIP preparation provides added beneficial glucose-lowering and insulinotropic actions over single incretins in mice with Type 2 diabetes and obesity

The present study examined the glucose-lowering and insulinotropic properties of acylated GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide) peptides in Type 2 diabetes and obesity. GLP-1, GIP, Liraglutide, N-AcGIP(Lys(37)Myr) (N-acetylGIP with myristic acid conjugated at Lys(37)), a simple combination of both peptides and a Lira-AcGIP preparation [overnight preparation of Liraglutide and N-AcGIP(Lys(37)Myr)] were incubated with DPP-IV (dipeptidyl peptidase-IV) to assess peptide stability, and BRIN-BD11 cells were used to evaluate cAMP production and insulin secretion. Acute glucose-lowering and insulinotropic actions were evaluated in Swiss TO mice. Subchronic studies on glucose homoeostasis, insulin secretion, food intake and bodyweight were evaluated in ob/ob mice. Liraglutide, N-AcGIP(Lys(37)Myr), a simple combination of both peptides and the Lira-AcGIP preparation demonstrated improved DPP-IV resistance (P<0.001), while stimulating cAMP production and insulin secretion (1.4-2-fold; P<0.001). The Lira-AcGIP preparation was more potent at lowering plasma glucose (20-51% reduction; P<0.05-P<0.001) and stimulating insulin secretion (1.5-1.8-fold; P<0.05-P<0.001) compared with Liraglutide and N-AcGIP(Lys(37)Myr) or a simple peptide combination. Daily administration of the Lira-AcGIP preparation to ob/ob mice lowered bodyweight (7-9%; P<0.05), food intake (23%; P<0.05) and plasma glucose (46% reduction; P<0.001), while increasing plasma insulin (1.5-1.6-fold; P<0.001). The Lira-AcGIP preparation enhanced glucose tolerance, insulin response to glucose and insulin content (P<0.05-P<0.001). These findings demonstrate that a combined preparation of the acylated GLP-1 and GIP peptides Liraglutide and N-AcGIP(Lys(37)Myr) markedly improved glucose-lowering and insulinotropic properties in diabetic obesity compared with either incretin mimetic given individually.

Inhibitors of the chymotrypsin-like activity of proteasome based on di- and tri-peptidyl α-keto aldehydes (glyoxals)

A series of peptidyl alpha-keto aldehydes (glyoxals) have been synthesised as putative inhibitors of the chymotryptic-like activity of proteasome. The most potent peptides, Cbz-Leu-Leu-Tyr-COCHO and Bz-Leu-Leu-Leu-COCHO, function as slow-binding reversible inhibitors, exhibiting final Ki values of approximately 3.0 nM. These are among the lowest values so far reported for (tri)peptide-based aldehyde-related inhibitors.

Novel Glucagon-Like Peptide-1 (GLP-1) Analog (Val8)GLP-1 Results in Significant Improvements of Glucose Tolerance and Pancreatic β-Cell Function after 3-Week Daily Administration in Obese Diabetic (ob/ob) Mice

This study evaluates the antidiabetic potential of an enzyme-resistant analog, (Val8)GLP-1. The effects of daily administration of a novel dipeptidyl peptidase IV-resistant glucagon-like peptide-1 (GLP-1) analog, (Val8)GLP-1, on glucose tolerance and pancreatic β-cell function were examined in obese-diabetic (ob/ob) mice. Acute intraperitoneal administration of (Val8)GLP-1 (6.25-25 nmol/kg) with glucose increased the insulin response and reduced the glycemic excursion in a dose-dependent manner. The effects of (Val8)GLP-1 were greater and longer lasting than native GLP-1. Once-daily subcutaneous administration of (Val8)GLP-1 (25 nmol/kg) for 21 days reduced plasma glucose concentrations, increased plasma insulin, and reduced body weight more than native GLP-1 without a significant change in daily food intake. Furthermore, (Val8)GLP-1 improved glucose tolerance, reduced the glycemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity. These effects were consistently greater with (Val8)GLP-1 than with native GLP-1, and both peptides retained or increased their acute efficacy compared with initial administration. (Val8)GLP-1 treatment increased average islet area 1.2-fold without changing the number of islets, resulting in an increased number of larger islets. These data demonstrate that (Val8)GLP-1 is more effective and longer acting than native GLP-1 in obese-diabetic ob/ob mice.

Antidiabetic potential of two novel fatty acid derivatised, N-terminally modified analogues of glucose-dependent insulinotropic polypeptide (GIP): N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37)

Abstract Fatty acid derivatisation was used to develop two novel, long-acting, N-terminally modified, glucose-dependent insulinotropic polypeptide (GIP) analogues, N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37). In contrast to GIP, which was rapidly degraded by in vitro incubation with dipeptidylpeptidase IV (DPP IV) (52% intact after 2 h), the analogues remained fully intact for up to 24 h. Both fatty acid-derivatised analogues stimulated cAMP production in GIP receptor Chinese hamster lung (CHL) fibroblasts (EC50 12.1–13.0 nM) and significantly improved in vitro insulin secretion from BRIN-BD11 cells (1.1- to 2.4-fold; p<0.05 to p<0.001) compared to control (5.6 mM glucose). Administration of N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) together with glucose in obese diabetic (ob/ob) mice significantly reduced the glycaemic excursion (1.4- and 1.5-fold, respectively; p<0.05 to p<0.01) and improved the insulinotropic response (1.5- and 2.3-fold, respectively; p<0.01 to p<0.001) compared to native peptide. Dose-response studies with N-AcGIP(LysPAL37) revealed that even the lowest concentration (6.25 nmol/kg) induced a highly significant decrease (1.4-fold; p<0.001) in the overall glycaemic excursion, coupled with a significant increase (2.0-fold; p<0.01) in circulating insulin. Furthermore, basal glucose values remained significantly reduced (p<0.05) and insulin values increased 24 h following a single injection of N-AcGIP(LysPAL37). The glucose-lowering action of the fatty acid-derivatised peptide was greater than that of N-AcGIP. These data demonstrate that novel fatty acid-derivatised analogues of N-terminally modified AcGIP function as long-acting GIP-receptor agonists, with significant antidiabetic potential.

Effects of short-term chemical ablation of the GIP receptor on insulin secretion, islet morphology and glucose homeostasis in mice

Abstract Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by endocrine K-cells in response to nutrient absorption. In this study we have utilized a specific and enzymatically stable GIP receptor antagonist, (Pro3)GIP, to evaluate the contribution of endogenous GIP to insulin secretion and glucose homeostasis in mice. Daily injection of (Pro3)GIP (25 nmol/kg body weight) for 11 days had no effect on food intake or body weight. Non-fasting plasma glucose concentrations were significantly raised (p<0.05) by day 11, while plasma insulin concentrations were not significantly different from saline treated controls. After 11 days, intraperitoneal glucose tolerance was significantly impaired in the (Pro3)GIP treated mice compared to control (p<0.01). Glucose-mediated insulin secretion was not significantly different between the two groups. Insulin sensitivity of 11-day (Pro3)GIP treated mice was slightly impaired 60 min post injection compared with controls. Following a 15 min refeeding period in 18 h fasted mice, food intake was not significantly different in (Pro3)GIP treated mice and controls. However, (Pro3)GIP treated mice displayed significantly elevated plasma glucose levels 30 and 60 min post feeding (p<0.05, in both cases). Postprandial insulin secretion was not significantly different and no changes in pancreatic insulin content or islet morphology were observed in (Pro3)GIP treated mice. The observed biological effects of (Pro3)GIP were reversed following cessation of treatment for 9 days. These data indicate that ablation of GIP signaling causes a readily reversible glucose intolerance without appreciable change of insulin secretion.

Novel GLP-1 analogue (Val8)GLP-1 results in significant improvements of glucose tolerance and pancreatic beta cell function after 3 weeks daily administration in obese diabetic (ob/ob) mice

This study evaluates the antidiabetic potential of an enzyme-resistant analog, (Val8)GLP-1. The effects of daily administration of a novel dipeptidyl peptidase IV-resistant glucagon-like peptide-1 (GLP-1) analog, (Val8)GLP-1, on glucose tolerance and pancreatic β-cell function were examined in obese-diabetic (ob/ob) mice. Acute intraperitoneal administration of (Val8)GLP-1 (6.25-25 nmol/kg) with glucose increased the insulin response and reduced the glycemic excursion in a dose-dependent manner. The effects of (Val8)GLP-1 were greater and longer lasting than native GLP-1. Once-daily subcutaneous administration of (Val8)GLP-1 (25 nmol/kg) for 21 days reduced plasma glucose concentrations, increased plasma insulin, and reduced body weight more than native GLP-1 without a significant change in daily food intake. Furthermore, (Val8)GLP-1 improved glucose tolerance, reduced the glycemic excursion after feeding, increased the plasma insulin response to glucose and feeding, and improved insulin sensitivity. These effects were consistently greater with (Val8)GLP-1 than with native GLP-1, and both peptides retained or increased their acute efficacy compared with initial administration. (Val8)GLP-1 treatment increased average islet area 1.2-fold without changing the number of islets, resulting in an increased number of larger islets. These data demonstrate that (Val8)GLP-1 is more effective and longer acting than native GLP-1 in obese-diabetic ob/ob mice.