Pro-carboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.
The protein glycogen phosphorylase has been linked to type 2 diabetes, indicating the importance of this target to human health. Hence, the search for potent and selective inhibitors of this enzyme, which may lead to antihyperglycaemic drugs, has received particular attention. Glycogen phosphorylase is a typical allosteric protein with five different ligand binding sites, thus offering multiple opportunities for modulation of enzyme activity. The present survey is focused on recent new molecules, potential inhibitors of the enzyme. The biological activity can be modified by these molecules through direct binding, allosteric effects or other structural changes. Progress in our understanding of the mechanism of action of these inhibitors has been made by the determination of high-resolution enzyme inhibitor structures (both muscle and liver). The knowledge of the three-dimensional structures of protein-ligand complexes allows analysis of how the ligands interact with the target and has the potential to facilitate structure-based drug design. In this review, the synthesis, structure determination and computational studies of the most recent inhibitors of glycogen phosphorylase at the different binding sites are presented and analyzed.
SummaryMannan, a polysaccharide isolated from yeast binds to C-type lectins of the mannose receptor family, expressed by antigen-presenting cells (APCs) including dendritic cells (DCs) and macrophages. As these receptors mediate endocytosis, they have been targeted with ligands to deliver antigens into APCs to initiate immune responses. Immunization with tumour antigen MUC1 conjugated to oxidized mannan (OM) or reduced mannan (RM) induced differential immune responses in mice, and only mice immunized with OM-MUC1 elicited strong MUC1-specific cytotoxic T lymphocyte responses and protected mice from a MUC1 tumour challenge. In this study, the adjuvant effect of mannan and its derivatives including OM and RM, in comparison to lipopolysaccharide, on DCs were investigated. Mannan, OM and RM were capable of stimulating mouse bone marrow-derived DC in vitro, eliciting enhanced allogeneic T-cell proliferation and enhancing OTI/OTII peptide-specific T-cell responses. Injection of mice with mannan, OM and RM induced a mature phenotype of lymph node and splenic DCs. Analysis by reverse transcription-polymerase chain reaction indicated that Manna, OM and RM also stimulated up-regulation of inflammatory cytokines including interleukin-1b and tumour necrosis factor-a, and differential T helper 1 (Th1)/Th2 cytokines. Subsequent experiments demonstrated that activation of DCs was Toll-like receptor-4-dependent. The data presented here, together with evidence reported previously on OM and RM in induction of immune responses in vivo, suggest that OM and RM exert a dual capacity to target antigen to APCs as well as mature DCs.
The presentation of peptides derived from tumor associated proteins (TAAs) by the major histocompatibility complex (MHC) to T cell receptor (TcR) initiates a cascade of events that constitute the immune response. Eliciting an effective immune response, however, requires the coordinated regulation of both the cellular and humoral arms of the immune system. The design of effective peptide-based vaccines for cancer immunotherapeutic applications, therefore, requires intimate knowledge and understanding of peptide-MHC (pMHC) as well as TcR-pMHC interactions. Despite the wealth of information available to date from X-ray crystallographic and biological studies, the task of rationally designing peptide-based vaccines that can effectively prevent and/or treat cancer cell proliferation remains challenging. The complexity of interactions involved are not readily predictable and are further complicated by the involvement of surrounding molecules in vivo, which can lead to reduced biological activity and/or unwanted side effects. Furthermore, the delivery of peptide-based vaccines into the cell, for further processing and presentation to effector cell, represents an additional challenge which needs to be addressed. The incorporation of appropriate chemical entities into peptide-based vaccines can improve cellular uptake thereby enhancing biological activity. Finally, the susceptibility of peptide-based vaccines to enzymatic degradation warrants the need for the incorporation of non-natural amino acids, retro-inversion and/or cyclization to improve bioavailability essentially reducing the required dosage with minimum side effects.
Immune responses to cancer cells can be elicited in vivo by administering synthetic peptides derived from proteins uniquely or overexpressed on tumor cells (tumor associated antigens--TAAs). Peptides derived from TAAs are presented in the context of major histocompatibility complex (MHC) molecules to cytotoxic T cells (CTL), which can recognize and lyze tumor cells. In contrast to peptides derived from an exogenous source (viral or bacterial), tumor peptides bind weakly to MHC class I molecules. The low binding affinity of these peptides makes them poor candidates for vaccination due to the poor immunogenic response produced. In order to enhance antigen recognition and hence immunogenicity, peptide binding affinity for MHC can be initially improved by modifying the "anchor" residues. However, the task at hand is highly unpredictable and minor changes in peptide sequence can alter/abolish the T cell response. Furthermore, despite the wealth of information obtained over the last decade from high resolution X-ray structures of MHC class I in complex with peptides (pMHC) as well as pMHC in complex with T cell receptor (TcR), prediction remains difficult. Nonetheless, peptides represent convenient chemical entities that can be rapidly synthesized in clinical grade for therapeutic applications. Herein, the rationale behind modifying TAAs will be discussed including the synthesis/use of proteolytically tolerant peptides (and peptide mimetics) which incorporate non-natural amino acids, retro-inversion and cyclization to improve bioavailability.
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