RESUMEN
Metalloproteases regulate a vast array of critical cellular processes such as proliferation, migration, repair, and invasion/metastasis. In so doing, metalloproteases have been shown to play key roles in the pathogenesis of multiple disorders including arteriosclerosis, arthritis, cancer metastasis, and ischemic brain injury. Therefore, much work has focused on developing metalloprotease inhibitors to provide a potential therapeutic benefit against the progression of these and other diseases. In order to produce a more potent inhibitor of metalloproteases, we synthesized multivalent displays of a metalloprotease inhibitor derived from the ring-opening metathesis polymerization (ROMP). Specifically, multivalent ligands of a broad-spectrum metalloprotease inhibitor, TAPI-2, were generated upon conjugation of the amine-bearing inhibitor with the ROMP-derived N-hydroxysuccinimide ester polymer. By monitoring the metalloprotease dependent cleavage of the transmembrane protein Semaphorin4D (Sema4D), we demonstrated an enhancement of inhibition by multivalent TAPI-2 compared to monovalent TAPI-2. To further optimize the potency of the multivalent inhibitor, we systematically varied the polymer length and inhibitor ligand density (mole fraction, χ). We observed that while ligand density plays a modest role in the potency of inhibition caused by the multivalent TAPI-2 display, the length of the polymer produces a much greater effect on inhibitor potency, with the shortest polymer achieving the greatest level of inhibition. These findings validate the use of multivalent display to enhance the potency of metalloprotease inhibitors and further, suggest this may be a useful approach to enhance potency of other small molecule towards their targets.
Asunto(s)
Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Metaloproteasas/antagonistas & inhibidores , Western Blotting , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Unión Proteica/efectos de los fármacos , Semaforinas/metabolismoRESUMEN
CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.
Asunto(s)
Linfocitos B/inmunología , Activación de Linfocitos/inmunología , Ácido N-Acetilneuramínico/química , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Secuencia de Carbohidratos , Línea Celular , Humanos , Ligandos , Datos de Secuencia Molecular , Lectina 2 Similar a Ig de Unión al Ácido Siálico/química , Transducción de SeñalRESUMEN
The applications of block copolymers are myriad, ranging from electronics to functionalized resins to therapeutics. The ring-opening metathesis polymerization (ROMP) is an especially valuable reaction for block copolymer assembly because each block can be generated with length control. We sought to use this polymerization to expand the repertoire of block copolymers by implementing a strategy that involves postpolymerization modification of a backbone bearing selectively reactive groups. To this end, we demonstrate that ROMP can be used to synthesize a block copolymer scaffold that possesses three types of functional groups-a succinimidyl ester, an alpha-chloroacetamide group, and a ketone-each of which can be modified independently. Thus, a single scaffold can be elaborated to afford a wide range of block copolymers. Exploiting this synthetic approach and the length control offered by ROMP, we assemble block copolymers capable of traversing the membrane and entering mammalian cells.
Asunto(s)
Polímeros/síntesis química , Acetamidas , Cetonas , Polímeros/química , SuccinimidasRESUMEN
Calls to bring interdisciplinary content and examples into introductory science courses have increased, yet strategies that involve course restructuring often suffer from the need for a significant faculty commitment to motivate change. Minimizing the need for dramatic course reorganization, the structure, reactivity, and chemical biology applications of classes of biological monomers and polymers have been integrated into introductory organic chemistry courses through three series of semester-long weekly assignments that explored (a) Carbohydrates and Oligosaccharides, (b) Amino Acids, Peptides, and Proteins, and (c) Nucleosides, Nucleotides, and Nucleic Acids. Comparisons of unannounced pre- and post tests revealed improved understanding of a reaction introduced in the assignments, and course examinations evaluated cumulative assignment topics. Course surveys revealed that demonstrating biologically relevant applications consistently throughout the semesters enhanced student interest in the connection between basic organic chemistry content and its application to new and unfamiliar bio-related examples. Covering basic material related to these classes of molecules outside of the classroom opened lecture time to allow the instructor to further build on information developed through the weekly assignments, teaching advanced topics and applications typically not covered in an introductory organic chemistry lecture course. Assignments were implemented as homework, either with or without accompanying discussion, in both laboratory and lecture organic courses within the context of the existing course structures.
Asunto(s)
Bioquímica/educación , Química Orgánica/educación , Enseñanza , Universidades , Aminoácidos/química , Carbohidratos/química , Evaluación Educacional , Humanos , Péptidos/química , Proteínas/química , EstudiantesRESUMEN
Benefits of incorporating research experiences into laboratory courses have been well documented, yet examples of research projects designed for the first semester introductory organic chemistry lab course are extremely rare. To address this deficiency, a Carbohydrate-Based human immunodeficiency virus (HIV) Inhibitor project consisting of a synthetic scheme of four reactions was developed for and implemented in the first semester organic lab. Students carried out the synthetic reactions during the last 6 of 10 total labs in the course, generating carbohydrate-based dimeric target molecules modeled after published dimers with application in HIV therapy. The project was designed to provide a research experience through use of literature procedures for reactions performed, exploration of variation in linker length in the target structure, and synthesis of compounds not previously reported in the scientific literature. Project assessment revealed strong student support, indicating enhanced engagement and interest in the course as a direct result of the use of scientific literature and the applications of the synthesized carbohydrate-based molecules. Regardless of discussed challenges in designing a research project for the first semester lab course, the finding from data analysis that a project implemented in the first semester lab had significantly greater student impact than a second semester project should provide motivation for development of additional research projects for a first semester organic course.
Asunto(s)
Fármacos Anti-VIH/química , Carbohidratos/química , Química Orgánica/educación , Biología Molecular/educación , Fármacos Anti-VIH/farmacología , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Carbohidratos/farmacología , Comprensión , Curriculum , Femenino , Infecciones por VIH/tratamiento farmacológico , Humanos , Laboratorios , Aprendizaje , Masculino , Investigación/educación , Estudiantes , Universidades , Adulto JovenRESUMEN
Modern research often requires collaboration of experts in fields, such as math, chemistry, biology, physics, and computer science to develop unique solutions to common problems. Traditional introductory undergraduate laboratory curricula in the sciences often do not emphasize connections possible between the various disciplines. We designed an interdisciplinary, medically relevant, project intended to help students see connections between chemistry and biology. Second term organic chemistry laboratory students designed and synthesized potential polymer inhibitors or inducers of polyglutamine protein aggregation. The use of novel target compounds added the uncertainty of scientific research to the project. Biology laboratory students then tested the novel potential pharmaceuticals in Huntington's disease model assays, using in vitro polyglutamine peptide aggregation and in vivo lethality studies in Drosophila. Students read articles from the primary literature describing the system from both chemical and biological perspectives. Assessment revealed that students emerged from both courses with a deeper understanding of the interdisciplinary nature of biology and chemistry and a heightened interest in basic research. The design of this collaborative project for introductory biology and organic chemistry labs demonstrated how the local interests and expertise at a university can be drawn from to create an effective way to integrate these introductory courses. Rather than simply presenting a series of experiments to be replicated, we hope that our efforts will inspire other scientists to think about how some aspect of authentic work can be brought into their own courses, and we also welcome additional collaborations to extend the scope of the scientific exploration.
Asunto(s)
Biología/educación , Química Orgánica/educación , Conducta Cooperativa , Investigación/educación , Evaluación Educacional/métodos , Humanos , Laboratorios , Aprendizaje , Estudiantes , UniversidadesRESUMEN
Multivalent interactions have been implicated in the binding of B-cell surface glycoprotein CD22 to its physiological ligands. Because CD22 can influence B-cell antigen receptor (BCR) signaling, multivalent ligands that cluster CD22 may influence B-cell responses. Here, we report an efficient synthesis of a fluorophore-labeled multivalent display of a CD22-binding trisaccharide, Neu5Acalpha2,6Galbeta1,4Glc, using the ring-opening metathesis polymerization (ROMP). Our synthetic strategy involves the modification of an N-hydroxysuccinimide (NHS) ester-substituted polymer generated by ROMP with the aminopropyl glycoside of the trisaccharide. The conjugation efficiency for the coupling is high; when 0.3 equiv of the trisaccharide derivative were used relative to NHS ester groups, the mole fraction (chi) of trisaccharide ligand incorporated onto the backbone was 0.3. A fluorescein-labeled version of the multivalent ligand binds to cells expressing CD22.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Moléculas de Adhesión Celular , Lectinas/metabolismo , Trisacáridos/síntesis química , Trisacáridos/metabolismo , Anticuerpos Monoclonales/química , Secuencia de Carbohidratos , Epítopos de Linfocito B/química , Epítopos de Linfocito B/metabolismo , Citometría de Flujo , Fluoresceína/química , Humanos , Células Jurkat/metabolismo , Ligandos , Linfoma de Células B/metabolismo , Datos de Secuencia Molecular , Ácido Peryódico/farmacología , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Células Tumorales CultivadasRESUMEN
Depending on the stimuli they encounter, B lymphocytes engage in signaling events that lead to immunity or tolerance. Both responses are mediated through antigen interactions with the B cell antigen receptor (BCR). Antigen valency is thought to be an important parameter in B cell signaling, but systematic studies are lacking. To explore this issue, we synthesized multivalent ligands of defined valencies using the ring-opening metathesis polymerization (ROMP). When mice are injected with multivalent antigens generated by ROMP, only those of high valencies elicit antibody production. These results indicate that ligands synthesized by ROMP can activate immune responses in vivo. All of the multivalent antigens tested activate signaling through the BCR. The ability of antigens to cluster the BCR, promote its localization to membrane microdomains, and augment intracellular Ca2+ concentration increases as a function of antigen valency. In contrast, no differences in BCR internalization were detected. Our results indicate that differences in the antigenicity of BCR ligands are related to their ability to elicit increases in intracellular Ca2+ concentration. Finally, we observed that unligated BCRs cluster with BCRs engaged by multivalent ligands, a result that suggests that signals mediated by the BCR are amplified through receptor arrays. Our data suggest a link between the mechanisms underlying signal initiation by receptors that must respond with high sensitivity.
Asunto(s)
Linfocitos B/fisiología , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/efectos de los fármacos , 2,4-Dinitrofenol/química , 2,4-Dinitrofenol/inmunología , Animales , Linfocitos B/inmunología , Haptenos , Ligandos , Ratones , Ratones Endogámicos BALB CRESUMEN
We report a general method for the solid-phase synthesis of polymers via the ring-opening metathesis polymerization (ROMP). The method involves polymerization in solution to form a block copolymer, immobilization of the polymer via reaction of one block with a resin-bound functional group, modification of the other block, and liberation of the polymer from the resin. We demonstrated the utility of this approach by generating a block copolymer with an N-hydroxysuccinimidyl ester-substituted block (for on-resin functionalization) and a maleimide-substituted block (for conjugation to the resin). We showed that the Diels-Alder reaction can be employed to immobilize the polymers and that amines of diverse structure can be used to modify the resin-bound polymers. The reversibility of the furan-maleimide Diels-Alder adduct was exploited to liberate the polymer from the support. Specifically, treatment of the resin with cyclopentadiene resulted in complete polymer release. The resulting polymers are functional: they were as potent in assays with the lectin concanavalin A as polymers generated by traditional solution routes. We anticipate that this method can be used for the rapid synthesis of diverse polymers via ROMP.
Asunto(s)
Polímeros/síntesis química , Ésteres/química , Inmovilización , Estructura MolecularRESUMEN
Compounds of the general structure A and B were investigated for their activity as lipoprotein(a), [Lp(a)], assembly (coupling) inhibitors. SAR around the amino acid derivatives (structure A) gave compound 14-6 as a potent coupling inhibitor. Oral dosing of compound 14-6 to Lp(a) transgenic mice and cymologous monkeys resulted in a>30% decrease in plasma Lp(a) levels after 1-2 weeks of treatment at 100 mg/kg/day.