Antibodies that catalyze the aldol reaction, a basic carbon-carbon bond-forming reaction, have been generated. The mechanism for antibody catalysis of this reaction mimics that used by natural class I aldolase enzymes. Immunization with a reactive compound covalently trapped a Lys residue in the binding pocket of the antibody by formation of a stable vinylogous amide. The reaction mechanism for the formation of the covalent antibody-hapten complex was recruited to catalyze the aldol reaction. The antibodies use the epsilon-amino group of Lys to form an enamine with ketone substrates and use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond. The antibodies control the diastereofacial selectivity of the reaction in both Cram-Felkin and anti-Cram-Felkin directions.
There is a pressing need for immunosuppressants with an improved safety profile. The search for novel approaches to blocking T-cell activation led to the development of the selective protein kinase C (PKC) inhibitor AEB071 (sotrastaurin). In cell-free kinase assays AEB071 inhibited PKC, with K i values in the subnanomolar to low nanomolar range. Upon T-cell stimulation, AEB071 markedly inhibited in situ PKC catalytic activity and selectively affected both the canonical nuclear factor-B and nuclear factor of activated T cells (but not activator protein-1) transactivation pathways. In primary human and mouse T cells, AEB071 treatment effectively abrogated at low nanomolar concentration markers of early T-cell activation, such as interleukin-2 secretion and CD25 expression. Accordingly, the CD3/CD28 antibody-and alloantigen-induced T-cell proliferation responses were potently inhibited by AEB071 in the absence of nonspecific antiproliferative effects. Unlike former PKC inhibitors, AEB071 did not enhance apoptosis of murine T-cell blasts in a model of activation-induced cell death. Furthermore, AEB071 markedly inhibited lymphocyte function-associated antigen-1-mediated T-cell adhesion at nanomolar concentrations. The mode of action of AEB071 is different from that of calcineurin inhibitors, and AEB071 and cyclosporine A seem to have complementary effects on T-cell signaling pathways.Phosphorylation of serine, threonine, and tyrosine residues is a primary mechanism for regulating protein function in eukaryotic cells. Protein kinases, the enzymes that catalyze these reactions, regulate essentially all cellular processes and have thus emerged as therapeutic targets for many human diseases. However, nearly all protein kinase inhibitors target the ATP binding site. For this reason, design of inhibitors that selectively target even a subset of the approximately 570 related human protein kinase domains continues to be a daunting challenge. Nevertheless, small-molecule inhibitors of Abelson tyrosine kinase and epidermal growth factor receptor have been recently developed into clinically useful anticancer drugs (for review, see Medinger and Drevs, 2005).The protein kinase C (PKC) family of serine/threonine kinases plays a central role in the adaptive immune system. PKC can be grouped into three categories according to the
The renin-anglotensin system (RAS) is the most important regulatory system of electrolyte homeostasis and blood pressure. We report here the development of transgenic rats carrying the human angiotensinogen TGR-(hAOGEN) and human renin TGR(hREN) genes. The plasma levels and tissue distribution of the transcription and translation products from both genes are described. A unique species specificity of the enzyme kinetics was observed. The human RAS components in the transgenic rats did not interact with the endogenous rat RAS in vivo. Insead, infusions of exogenous human RAS components specifically interacted with human transgene translation products. Thus, iion of human renin in TGR(hAOGEN) led to an increase of angioensin H and an elevation of blood pressure, which could not be antagonized by the human-specific renin enzyme inhibitor Ro 42-5892. Rat renin also elevated blood pressure and angiotensin H in TGR(hAOGEN); however, this effect was not antagonized by the human renin inhibitor. Compared to mice, rats offer the advantage of chronic instrumentation and repetitive, sophisticated, hemodynamic, and endocrinological investigations. Thus, transgenic rat models with human-specific enzyme kinetics permit primate-specific analyses in non-primate in vivo and in vitro experimental systems.The successful incorporation of renin-angiotensin (RAS) genes into transgenic mice has been reported (1, 2). Rats, on the other hand, present specific technical problems with respect to the generation of transgenic animals. The transgenic rats TGR(mREN2)27, which harbor a mouse renin gene and exhibit fulminant hypertension, provided evidence for a monogenetic form of hypertension (3).Transgenic mice harboring the genes of mouse renin and angiotensinogen (2), rat renin and angiotensinogen (4), as well as human renin (1) have been described. Thus far, most ofthe RAS gene constructs used for the generation of transgenic animals interacted directly with the host RAS (2, 4).Since the genetic background for the transgenes appears to be of primary importance for the development of hypertension (5), we developed transgenic rats harboring the complete human renin TGR(hREN) and human angiotensinogen TGR(hAOGEN) genes. These rats allow studies into the regulation of human genes in non-primate animal models (6). In vitro, the species specificity of human renin and angiotensinogen is well known. Transgenic animals permit the study of specific interactions with the human transgene products in vivo, without interference from the host RAS.EXPERIMENTAL PROCEDURES Transgenic Techniques. Linear DNA fiagments consisting of either the entire human renin gene (1, 7) or the entire human angiotensinogen gene (8), as described elsewhere (1, 3), were injected into the male pronuclei offertilized, outbred Sprague-Dawley (SD) rat eggs.Immnnalyses of Renin and en. Human renin was immunologically measured in transgenic rat plasma. The direct and specific measurement of human renin used two pairs of monoclonal antibodies in an immunoradiometric assay. ...
Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC(50) = 6.9 +/- 0.9 nM) but is structurally different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the C(26)=C(27) exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity of 2 for cyclophilin (IC(50) = 29 +/- 2.1 nM) is essentially identical to SFA, which indicates that the interaction between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This observation was confirmed by the X-ray crystal structure resolved at 2.1 A of cyclophilin A complexed to macrolide 16, a close analogue of 2. The X-ray crystal structure showed that macrolide 16 binds to the same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure-activity relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or (2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide unit, which together provide an extremely strong affinity for cyclophilin A.
Chronic allograft nephropathy is characterized by chronic inflammation and fibrosis. Because retinoids exhibit anti-proliferative, anti-inflammatory, and anti-fibrotic functions, the effects of low and high doses of 13-cis-retinoic acid (13cRA) were studied in a chronic Fisher3443 Lewis transplantation model. In 13cRA animals, independent of dose (2 or 20 mg/kg body weight/day) and start (0 or 14 days after transplantation) of 13cRA administration, serum creatinine was significantly lower and chronic rejection damage was dramatically reduced, including subendothelial fibrosis of preglomerular vessels and chronic tubulointerstitial damage. The number of infiltrating mononuclear cells and their proliferative activity were significantly diminished. The mRNA expression of chemokines (MCP-1/CCL2, MIP-1␣/CCL3, IP-10/CXCL10, RANTES/CCL5) and proteins associated with fibrosis (plasminogen activator inhibitor-1, transforming growth factor-1, and collagens I and III) were strikingly lower in treated allografts. In vitro, activated peritoneal macrophages of 13cRA-treated rats showed a pronounced decrease in protein secretion of inflammatory cytokines (eg, tumor necrosis factor-␣, interleukin-6). The suppression of the proinflammatory chemokine RANTES/CCL5 ؋ 13cRA in fibroblasts could be mapped to a promoter module comprising IRF-1 and nuclear factor-B binding elements, but direct binding of retinoid receptors to promoter elements could be excluded. In summary, 13cRA acted as a potent immunosuppressive and antifibrotic agent able to prevent and inhibit progression of chronic allograft nephropathy.
A series of novel maleimide-based inhibitors of protein kinase C (PKC) were designed, synthesized, and evaluated. AEB071 (1) was found to be a potent, selective inhibitor of classical and novel PKC isotypes. 1 is a highly efficient immunomodulator, acting via inhibition of early T cell activation. The binding mode of maleimides to PKCs, proposed by molecular modeling, was confirmed by X-ray analysis of 1 bound in the active site of PKCalpha.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.