Peter C. Tyler

Liposomal delivery of antigen to human dendritic cells

This study investigated whether formulation of antigen in mannosylated liposomes enhanced uptake and activation of dendritic cells (DC) and increased the ability of DC to induce primed T cell proliferation compared to formulation of antigen in unmodified liposomes or in solution. Immature human DC were generated from peripheral blood monocytes cultured with GM-CSF and IL-4. Uptake of antigen by DC and the degree of expression of the cell surface markers MHC class II, CD80, CD86 and the DC maturation marker CD83, was investigated by flow cytometry following incubation with liposomes or solution containing FITC-conjugated antigen. Exposure to liposomes containing FITC-ovalbumin resulted in enhanced expression of cell surface markers when compared to exposure to antigen in solution. Expression was highest following exposure to mannosylated liposomes. Mannosylated liposomes containing tetanus toxoid (TT) stimulated primed T cell proliferation more effectively than TT-neutral liposomes or TT-solution. This work suggests that mannosylated liposomes provide a versatile delivery vehicle for initiating enhanced immune responses to encapsulated peptide or protein vaccines.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes.

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

Transition-state analogs as inhibitors of human and malarial hypoxanthine-guanine phosphoribosyltransferases

The proposed transition state for hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) has been used to design and synthesize powerful inhibitors that contain features of the transition state. The iminoribitols (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinHP) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol 5-phosphate (immucillinGP) are the most powerful inhibitors yet reported for both human and malarial HGPRTs. Equilibrium binding constants are >1,000-fold tighter than the binding of the nucleotide substrate. The NMR spectrum of malaria HGXPRT in the Michaelis complex reveals downfield hydrogen-bonded protons. The chemical shifts move farther downfield with bound inhibitor. The inhibitors are lead compounds for species-specific antibiotics against parasitic protozoa. The high-resolution crystal structure of human HGPRT with immucillinGP is reported in the companion paper.

Third-Generation Immucillins: Syntheses and Bioactivities of Acyclic Immucillin Inhibitors of Human Purine Nucleoside Phosphorylase

ImmH (1) and DADMe-ImmH (2) are potent inhibitors of human purine nucleoside phoshorylase (PNP), developed by us and currently in clinical trials for the treatment of a variety of T-cell related diseases. Compounds 1 and 2 were used as templates for the design and synthesis of a series of acyclic immucillin analogues (8-38) in order to identify simplified alternatives to 1 and 2. SerMe-ImmG (8) and DATMe-ImmG (9) displayed the lowest inhibition constants of 2.1 and 3.4 pM, respectively, vs PNP. It was postulated that the flexible natures of 8 and 9 enabled them to adopt conformations resembling those of 1 and 2 within the active site of PNP and that the positioning of two hydroxyl groups was critical for picomolar activity. SerMe-ImmH (10, K(d) = 5.2 pM) was shown to be orally available in mice with a long biological residence time on blood PNP.

Synthesis of transition state inhibitors for N-riboside hydrolases and transferases

Abstract A number of 1,4-dideoxy-1,4-imino-1-(S)-(substituted phenyl)- d -ribitols bearing aromatic OH, NH2, NO2, CO2H and halogeno moieties, and a 3-pyridyl analogue have been synthesized. The key step is the condensation of aryllithium or aryl Grignard reagents with the imine 3; derived from the protected 1,4-dideoxy-1,4-imino- d -ribitol 4.

Synthesis of a Targeted Library of Heparan Sulfate Hexa- to Dodecasaccharides as Inhibitors of β-Secretase: Potential Therapeutics for Alzheimer’s Disease

Heparan sulfates (HS) are a class of sulfated polysaccharides that function as dynamic biological regulators of the functions of diverse proteins. The structural basis of these interactions, however, remains elusive, and chemical synthesis of defined structures represents a challenging but powerful approach for unravelling the structure-activity relationships of their complex sulfation patterns. HS has been shown to function as an inhibitor of the β-site cleaving enzyme β-secretase (BACE1), a protease responsible for generating the toxic Aβ peptides that accumulate in Alzheimers disease (AD), with 6-O-sulfation identified as a key requirement. Here, we demonstrate a novel generic synthetic approach to HS oligosaccharides applied to production of a library of 16 hexa- to dodecasaccharides targeted at BACE1 inhibition. Screening of this library provided new insights into structure-activity relationships for optimal BACE1 inhibition, and yielded a number of potent non-anticoagulant BACE1 inhibitors with potential for development as leads for treatment of AD through lowering of Aβ peptide levels.

Synthesis of Transition State Analogue Inhibitors for Purine Nucleoside Phosphorylase and N-Riboside Hydrolases

Abstract Syntheses of the ‘Immucillins’, potent aza-C-nucleoside inhibitors of purine nucleoside phosphorylase are reported as well as those of 5-deoxy-, 5-deoxyfluoro- and 2-deoxy- analogues and others having modified bases.

Azetidine based transition state analogue inhibitors of N-ribosyl hydrolases and phosphorylases.

N-ribosyl phosphorylases and hydrolases catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon from the fixed purine to phosphate and water nucleophiles, respectively. As the lysis reaction progresses along the reaction coordinate, the distance between the purine and carbocation increases and the distance between carbocation and nucleophile decreases. Immucillin-H and DADMe-immucillin-H have been shown previously to be potent inhibitors of purine nucleoside phosphorylases and lie more toward the reactant and products side of this reaction coordinate, respectively. Both these enzyme inhibitors, which are currently in human clinical trials for different indications, are chiral and expensive to manufacture. We now report the synthesis of azetidine analogues of the DADMe-immucillins, which, despite their lack of stereochemical complexity, remain potent inhibitors (equilibrium dissociation constants as low as 229 pM) of purine nucleoside phosphorylase (PNP), methylthioadenosine phosphorylase (MTAP), and methylthioadenosine nucleosidase (MTAN), with potential utility as drug candidates.

Mannosylated liposomes as antigen delivery vehicles for targeting to dendritic cells

The immune stimulating ability of mannosylated liposomes containing FITC‐ovalbumin as a model antigen and displaying either a branched tri‐mannose or a mono‐mannose ligand on the liposome surface was investigated in human monocyte‐derived dendritic cells (MoDCs) and murine bone‐marrow‐derived dendritic cells (BMDCs). Uptake of liposomes, dendritic cell activation and proliferation of CD8+ T cells from OT‐I transgenic mice were determined by flow cytometry. Uptake of liposomes displaying the tri‐mannose ligand was enhanced in human MoDCs compared with both non‐mannosylated liposomes and liposomes displaying mono‐mannose ligands. However, this increased uptake did not result in an increase in expression of CD80 or CD86 on the surface of the MoDCs. In contrast, neither tri‐mannose‐ nor mono‐mannose‐containing liposomes were taken up by murine BMDCs to a greater extent than non‐mannose‐containing liposomes. The expression of CD86 and CD40 on the surface of BMDCs was not increased after exposure to mannosylated lipo‐somes and BMDCs incubated with mannosylated liposomes were not able to stimulate proliferation of CD8+ T cells to any greater extent than BMDCs incubated with non‐mannosylated liposomes. These findings suggest that while mannose‐containing ligands can enhance the uptake of antigen‐containing liposomes by some dendritic cells, important differences in the affinity of carbohydrate‐binding receptors for mannose‐containing ligands do exist between species. In addition, the increase in uptake of antigen by dendritic cells using mannosylated liposomes does not necessarily result in enhanced dendritic cell activation.

Four generations of transition-state analogues for human purine nucleoside phosphorylase

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (, first-generation) contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (, second-generation), uses a methylene-bridged dihydroxypyrrolidine cation with two asymmetric centers. DATMe-Immucillin-H (, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5′-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.