Scott A. Mitchell

Improved bioavailability to the brain of glycosylated Met-enkephalin analogs

The blood-brain barrier prevents the entry of many potentially therapeutic peptide drugs to the brain. Glycosylation has shown potential as a methodology for improving delivery to the CNS. Previous studies have shown improved bioavailability and improved centrally mediated analgesia of glycosylated opioids. In this study we investigate the effect of glycosylation on the cyclic opioid peptide [D-Cys(2,5),Ser(6),Gly(7)] enkephalin. The peptide was glycosylated on the Ser(6) via an O-linkage with various sugar moieties and alignments. The peptides were then investigated for receptor binding, physiochemical attributes, in situ brain uptake in female Sprague-Dawley rats and antinociception in male ICR mice. Glycosylation resulted in a slight decrease in affinity to the delta-opioid receptor, and mixed effect on binding to the mu-opioid receptor. There was a significant decrease in lipophilicity resulting from glycosylation and a slight reduction in binding to bovine serum albumin. In situ perfusion showed that brain uptake was improved by up to 98% for several of the glycosylated peptides, and the nociceptive profiles of the peptides, in general, followed the rank order of peptide entry to the brain with up to a 39-fold increase in A.U.C.

Discovery of GS-9973, a Selective and Orally Efficacious Inhibitor of Spleen Tyrosine Kinase.

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune, inflammatory, and oncology disease indications. The most advanced Syk inhibitor, R406, 1 (or its prodrug form fostamatinib, 2), has shown efficacy in multiple therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed, at least in part, to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973, 68, a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications.

Enkephalin-based drug design: conformational analysis of O-linked glycopeptides by NMR and molecular modeling

Abstract Glycosylation provides an effective means of enhancing penetration of the blood–brain barrier by pharmacologically active peptides. Glycosylated enkephalin analogues demonstrate much greater analgesic effects than their unglycosylated counterparts when administered peripherally. The solution conformations of glycopeptide enkephalin analogues with the sequences H-Tyr-c-[ d -Cys-Gly-Phe- d -Cys]-Ser(β-O-Glcp)-Gly-NH2, 2, and H-Tyr-c-[ d -Cys-Gly-Phe- d -Cys]-Ser(α-O-Glcp)-Gly-NH2, 3, have been determined by NMR and molecular modeling, and were compared to the unglycosylated peptide H-Tyr-c-[ d -Cys-Gly-Phe- d -Cys]-Ser-Gly-NH2, 1, to determine the impact of glycosylation on peptide conformation. The only observed conformational effects were on the residue of attachment, Ser6, and on the adjacent Gly7-amide. This has important implications in peptide-based drug design in that strategically placed glycosylation can improve transport without destruction of the receptor selectivity of a pre-existing non-glycosylated peptide pharmacophore.

FRI0049 Preclinical Characterization of GS-9876, A Novel, Oral SYK Inhibitor That Shows Efficacy in Multiple Established Rat Models of Collagen-Induced Arthritis

Background Spleen Tyrosine Kinase (SYK) mediates signaling in a range of hematopoietic cells involved in the initiation and progression of RA including B cells, monocytes, macrophages, dendritic cells, and osteoclasts. There is strong preclinical validation for SYK as a therapeutic target for RA based on cellular data and animal models of disease. We have identified GS-9876 as a potent and selective SYK inhibitor with pharmaceutical properties compatible with once daily oral dosing in human. Objectives To characterize the cellular activity of GS-9876 on pathologically relevant pathways in RA, and establish target inhibition requireements for efficacy in rat models of arthritis. Methods The potency and selectivity of GS-9876 were characterized in biochemical and cellular assays. Effects in B cells were measured by inhibition of BCR-mediated protein phosphorylation, CD69 and CD86 expression, and in macrophages by inhibition of immune-complex (IC)-stimulated cytokine release. Cellular selectivity was demonstrated by comparing the inhibition of B cell proliferation versus T cell proliferation. GS-9876 potency in human blood was evaluated by inhibition of phosphorylated SYK (pSYK), CD63 expression on basophils, and CD69 expression on B cells. The in vivo efficacy of GS-9876 was tested in rat models of collagen-induced arthritis (CIA). Results GS-9876 is a potent SYK inhibitor (IC50=9.5±4.3 nM) and is highly selective against a panel of 456 other kinases. GS-9876 inhibited anti-IgM stimulated phosphorylation of AKT, BLNK, BTK, ERK, MEK, and PKCδ in human B cells with EC50 values of 24–51 nM. Functionally, GS-9876 inhibited anti-IgM mediated CD69 and CD86 expression on B-cells (EC50=112±10 nM and 164±15 nM, respectively) and anti-IgM /anti-CD40 co-stimulated B cell proliferation (EC50=108±55 nM). In human macrophages, GS-9876 inhibited IC-stimulated TNFα and IL-1β release (EC50=121±77 nM and 9±17 nM, respectively). Anti-CD3/anti-CD28 stimulated T cell proliferation was weakly inhibited (EC50=1291±398 nM), with selectivity >10-fold versus the inhibition of B cell proliferation. In human blood, GS-9876 blocked SYK phosphorylation, CD69 expression on B cells, and CD63 expression in basophils. GS-9876 demonstrated a dose-dependent improvement in clinical score and histopathology parameters with once-daily dosing in short and long term rat CIA models. Significant efficacy could be achieved with GS-9876 doses that produced trough pSYK inhibition of <50%. Conclusions GS-9876 is a novel SYK inhibitor that potently inhibits multiple cellular events implicated in RA pathogenesis and displays excellent in vivo efficacy in rat CIA models after once-daily dosing. GS-9876 has markedly improved selectivity over competitor SYK programs. Our data support the development of GS-9876 in inflammatory diseases, with potential for an improved safety profile. Disclosure of Interest J. Di Paolo Shareholder of: Gilead Sciences, P. Blomgren Shareholder of: Gilead Sciences, M. Dolton Shareholder of: Gilead Sciences, R. Jones Shareholder of: Gilead Sciences, J. Kropf Shareholder of: Gilead Sciences, T. Lee Shareholder of: Gilead Sciences, S. Mitchell Shareholder of: Gilead Sciences, B. Murray Shareholder of: Gilead Sciences, K. Suekawa-Pirrone Shareholder of: Gilead Sciences, S. Wise Shareholder of: Gilead Sciences, J. Xu Shareholder of: Gilead Sciences, Z. Zhao Shareholder of: Gilead Sciences, K. Currie Shareholder of: Gilead Sciences

Synthesis of several O-glycopeptide analogs of enkephalin

Work within the Polt laboratory has focussed on the exploration and exploitation of carbohydrate moieties in the design and synthesis of opiate glycopeptides. These glycopeptides have been synthesized based on the pharmacophore D-Cys -enkephalin (DCDCE) [1]. A -D-glucose-containing analog of DCDCE, LSZ-1025, displayed potent analgesia when administered peripherally [2,3]. In an effort to understand the role of the amino acid glycoside in both penetration of the blood-brain barrier (BBB) and opiate receptor binding and stimulation, a series of glycopeptides were synthesized. This work reflects the optimized synthesis of several enkephalin glycopeptides, represented by several structural modifications. Incorporation of D-amino acid glucosides to test the environment of the amino acid-glycoside linkage, variance of the glycan moiety on serine to verify the impact of different sugars, and implementation of several different pharmacophores, such as in SAM-1095 where a linear pharmacophore [4] was used, highlight our design rationale. The impact of these changes was assessed after detailed pharmacological testing.

Practical glycopeptide analgesics: Blood-brain barrier transport and binding of glycosylated enkephalin analogs

Robin Polt, Richard D. Egleton, Edward J. Bilsky, Scott A. Mitchell, Caroline T. Kriss, Matt R. Pratt, Peg Davis, Heather Jones, Frank Porrecca, Henry I. Yamamura, and Victor J. Hruby Department of Chemistry, University of Arizona, Tucson, AZ 85721, U.S.A.; Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724, U.S.A.; and Department of Biological Sciences, University of Northern Colorado, CO 80639, U.S.A.

Chemical Biology and Biomedicine: Enkephalin-Derived Glycopeptide Analgesics

Pain relief has been sought since the earliest humans gathered plants for various poultices and potions. Alchemists, shamans, and contemporary consumers alike have all shared the desire for a compound or a biological preparation that alleviates pain. Hippocrates, ca. 500 BC, reported that ingestion of the broth of boiled willow tree bark (genus Salix) relieved pain. Over two millennia later, the pharmacologically active compounds in the bark were isolated and characterized. Laroux et al. isolated the analgesic salicin in 1829, and Piria et al. isolated salicylic acid in 1840. The first non-narcotic pharmaceutical for pain relief was Von Gerhardt’s acetylsalicylic acid (aspirin) in 1853, which was commercialized by Felix Hofmann at Bayer in 1893 [1]. Since then many peripheral (non-narcotic) analgesics have been developed and commercialized (Fig. 1).