Antiangiogenic Activity and in Silico Cereblon Binding Analysis of Novel Thalidomide Analogs

Funding: This research was supported in part by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute (ZIA SC006538); in part with Federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E; the Intramural Research Program of the National Institute on Aging, National Institutes of Health; and a Wellcome Trust-NIH PhD Studentship to SB, WDF, and NV (Grant number 098252/Z/12/Z). Acknowledgments: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.Peer reviewedPublisher PD

Antiangiogenic Activity and in Silico Cereblon Binding Analysis of Novel Thalidomide Analogs

Due to its antiangiogenic and anti-immunomodulatory activity, thalidomide continues to be of clinical interest despite its teratogenic actions, and efforts to synthesize safer, clinically active thalidomide analogs are continually underway. In this study, a cohort of 27 chemically diverse thalidomide analogs was evaluated for antiangiogenic activity in an ex vivo rat aorta ring assay. The protein cereblon has been identified as the target for thalidomide, and in silico pharmacophore analysis and molecular docking with a crystal structure of human cereblon were used to investigate the cereblon binding abilities of the thalidomide analogs. The results suggest that not all antiangiogenic thalidomide analogs can bind cereblon, and multiple targets and mechanisms of action may be involved

Molecular modeling of the bacterial chemotaxis receptors Tar and Trg

Thesis (Ph. D.)--University of Washington, 2001Bacterial chemotaxis receptors signal across the membrane by a conformational change that traverses their periplasmic, transmembrane, and cytoplasmic domains. The mechanism of this conformational change is controversial and is not completely understood. Molecular models of the periplasmic and transmembrane domains of the homodimeric chemotaxis receptors Trg and Tar were constructed, using the coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of the periplasmic domain of Tar to establish register and positioning. The models were tested and refined using the extensive experimental data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices.These refined models were used to assess the effects of inter-helical disulfides, using a measure of disulfide potential energy, on two previously proposed mechanisms for ligand-induced conformational changes in the receptor structure: an axial sliding motion of one helix in a subunit, and a rotational motion between the two subunits in the dimer. These proposed mechanisms were tested further, along with the theory that receptor signaling might involve a change in dynamics, using molecular dynamics simulations of the fully solvated periplasmic and transmembrane domains.Testing of disulfide effects on receptor motion showed that a sliding motion of transmembrane helix 2 is consistent with experimental data on receptor signaling. However, inter-helical disulfides would not significantly constrain an inter-subunit rotational motion. The molecular dynamics simulations showed that the transmembrane domain has a high degree of dynamic flexibility, and that in the isolated periplasmic domain ligand binding induces an inter-subunit rotation that is of much greater magnitude than was previously concluded from analysis of the periplasmic domain crystal structures.These results suggest a new model for transmembrane signaling in which an intersubunit rotational motion is converted into a helical sliding motion via a flexible transmembrane domain and linker region. Thus, both proposed mechanisms occur and have functional roles

Synthesis of seco-B-Ring Bryostatin Analogue WN‑1 via C−C Bond- Forming Hydrogenation: Critical Contribution of the B‑Ring in Determining Bryostatin-like and Phorbol 12-Myristate 13-Acetate- like Properties

*S Supporting Information ABSTRACT: The seco-B-ring bryostatin analogue, macrodiolide WN-1, was prepared in 17 steps (longest linear sequence) and 30 total steps with three bonds formed via hydrogen-mediated C−C coupling. This synthetic route features a palladium-catalyzed alkoxycarbonylation of a C2-symmetric diol to form the C9-deoxygenated bryostatin A-ring. WN-1 binds to PKCα (Ki = 16.1 nM) and inhibits the growth of multiple leukemia cell lines. Although structural features of the WN-1 A-ring and C-ring are shared by analogues that display bryostatin-like behavior, WN-1 displays PMA-like behavior in U937 cell attachment and proliferation assays, as well as in K562 and MV-4-11 proliferation assays. Molecular modeling studies suggest the pattern of internal hydrogen bonds evident in bryostatin 1 is preserved in WN-1, and that upon docking WN-1 into the crystal structure of the C1b domain of PKCδ, the binding mode of bryostatin 1 is reproduced. The collective data emphasize the critical contribution of the B-ring to the function of the upper portion of the molecule in conferring a bryostatin-like pattern of biological activity. The bryostatins are a family of marine macrolides isolated by Pettit and co-workers from the bryozoan Bugula neritina based on an assay of their anti-neoplastic activity against the P388 leukemia cell system.1 Bryostatin 1 (Figure 1), the mos

Therapeutic Antibodies to Ganglioside GD2 Evolved from Highly Selective Germline Antibodies

Antibodies play a crucial role in host defense and are indispensable research tools, diagnostics, and therapeutics. Antibody generation involves binding of genomically encoded germline antibodies followed by somatic hypermutation and in vivo selection to obtain antibodies with high affinity and selectivity. Understanding this process is critical for developing monoclonal antibodies, designing effective vaccines, and understanding autoantibody formation. Prior studies have found that antibodies to haptens, peptides, and proteins evolve from polyspecific germline antibodies. The immunological evolution of antibodies to mammalian glycans has not been studied. Using glycan microarrays, protein microarrays, cell binding studies, and molecular modeling, we demonstrate that therapeutic antibodies to the tumor-associated ganglioside GD2 evolved from highly specific germline precursors. The results have important implications for developing vaccines and monoclonal antibodies that target carbohydrate antigens. In addition, they demonstrate an alternative pathway for antibody evolution within the immune system that is distinct from the polyspecific germline pathway

Diagnostic cross-linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identity

Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor TrgE from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the TrgE domain was created using the homodimeric, four-helix bundle structure of the TarS domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the TrgE periplasmic domain is very similar to the TarS domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models