Structural Studies of Wnts and Identification of an LRP6 Binding Site

SummaryWnts are secreted growth factors that have critical roles in cell fate determination and stem cell renewal. The Wnt/β-catenin pathway is initiated by binding of a Wnt protein to a Frizzled (Fzd) receptor and a coreceptor, LDL receptor-related protein 5 or 6 (LRP5/6). We report the 2.1 Å resolution crystal structure of a Drosophila WntD fragment encompassing the N-terminal domain and the linker that connects it to the C-terminal domain. Differences in the structures of WntD and Xenopus Wnt8, including the positions of a receptor-binding β hairpin and a large solvent-filled cavity in the helical core, indicate conformational plasticity in the N-terminal domain that may be important for Wnt-Frizzled specificity. Structure-based mutational analysis of mouse Wnt3a shows that the linker between the N- and C-terminal domains is required for LRP6 binding. These findings provide important insights into Wnt function and evolution

Iterative design and optimization of initially inactive Proteolysis Targeting Chimeras (PROTACs) identify VZ185 as a potent, fast and selective von Hippel-Lindau (VHL)-based dual degrader probe of BRD9 and BRD7

Developing PROTACs to redirect the ubiquitination activity of E3 ligases and potently degrade a target protein within cells can be a lengthy and unpredictable process, and it remains unclear whether any combination of E3 and target might be productive for degradation. We describe a probe-quality degrader for a ligase-target pair deemed unsuitable: the von Hippel-Lindau (VHL) and BRD9, a bromodomain-containing subunit of the SWI/SNF chromatin remodeling complex BAF. VHL-based degraders could be optimized from suboptimal compounds in two rounds by systematically varying conjugation patterns and linkers, and monitoring cellular degradation activities, kinetic profiles, and ubiquitination, as well as ternary complex formation thermodynamics. The emerged structure-activity relationships guided the discovery of VZ185, a potent, fast and selective degrader of BRD9 and of its close homolog BRD7. Our findings qualify a new chemical tool for BRD7/9 knockdown, and provide a roadmap for PROTAC development against seemingly incompatible target-ligase combinations

Estudos visando a síntese de compostos macrocíclicos e heterocíclicos via reações multicomponentes de Ugi

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2017.De modo geral, estruturas cíclicas ocupam um lugar de destaque na química orgânica e rotas sintéticas para a obtenção desta classe de moléculas são altamente desejáveis. É crescente o número de publicações em que reações multicomponente de Ugi são utilizadas visando à síntese de compostos macrocíclicos e heterocíclicos. Desta forma, neste trabalho foram propostas duas estratégias sintéticas distintas, ambas baseadas na reação de Ugi, para a construção de um acervo de compostos cíclicos. A primeira delas, baseada nos preceitos da MiBs (Multiple Multicomponent Macrocyclizations Including Bifunctional Building Blocks), em que componentes bifuncionais são necessários, visou a síntese de compostos macrocíclicos via reações consecutivas de Ugi empregando diácidos carboxílicos preparados a partir de reações de Ugi do tipo U-5C-4CR. A segunda estratégia buscou empregar um aldeído polifuncionalizado, produto de um rearranjo de Claisen em adutos de Morita-Baylis-Hillman, como um dos componentes em reações multicomponentes de Ugi. Esta abordagem permitiu a obtenção de compostos heterocíclicos estruturalmente distintos pela utilização de diferentes estratégias de ciclização.In general, cyclic structures occupy a prominent place in organic chemistry and synthetic routes for obtaining this class of molecules are highly desirable. There is a growing number of publications in which Ugi multi-component reactions are used for the synthesis of macrocyclic and heterocyclic compounds. In this work, two different synthetic strategies were proposed, both based on the Ugi reaction, for the construction of a collection of cyclic compounds. The first one, based on the precepts of the MiBs (Multiple Multicomponent Macrocyclics Including Bifunctional Building Blocks), in which bifunctional components are required, aimed the synthesis of macrocyclic compounds via consecutive Ugi reactions using carboxylic diacids prepared from U- 5C-4CR Ugi variant. The second strategy employ a polyfunctionalized aldehyde, product of a Claisen rearrangement in Morita-Baylis-Hillman adducts, as one of the components in Ugi multicomponent reactions. This approach allowed structurally distinct heterocyclic compounds readly obtained by the use of different cyclization strategies

9H-purine scaffold reveals induced-fit pocket plasticity of the BRD9 bromodomain

The 2-amine-9H-purine scaffold was identified as a weak bromodomain template and was developed via iterative structure based design into a potent nanomolar ligand for the bromodomain of human BRD9 with small residual micromolar affinity toward the bromodomain of BRD4. Binding of the lead compound 11 to the bromodomain of BRD9 results in an unprecedented rearrangement of residues forming the acetyllysine recognition site, affecting plasticity of the protein in an induced-fit pocket. The compound does not exhibit any cytotoxic effect in HEK293 cells and displaces the BRD9 bromodomain from chromatin in bioluminescence proximity assays without affecting the BRD4/histone complex. The 2-amine-9H-purine scaffold represents a novel template that can be further modified to yield highly potent and selective tool compounds to interrogate the biological role of BRD9 in diverse cellular systems

The TIP60 complex is a conserved coactivator of HIF1A

Hypoxia-inducible factors (HIFs) are critical regulators of the cellular response to hypoxia. Despite their established roles in normal physiology and numerous pathologies, the molecular mechanisms by which they control gene expression remain poorly understood. We report here a conserved role for the TIP60 complex as a HIF1 transcriptional cofactor in Drosophila and human cells. TIP60 (KAT5) is required for HIF1-dependent gene expression in fly cells and embryos and colorectal cancer cells. HIF1A interacts with and recruits TIP60 to chromatin. TIP60 is dispensable for HIF1A association with its target genes but is required for HIF1A-dependent chromatin modification and RNA polymerase II activation in hypoxia. In human cells, global analysis of HIF1A-dependent gene activity reveals that most HIF1A targets require either TIP60, the CDK8-Mediator complex, or both as coactivators for full expression in hypoxia. Thus, HIF1A employs functionally diverse cofactors to regulate different subsets of genes within its transcriptional program.Fil: Perez Perri, Joel Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Dengler, Verónica L.. Howard Hughes Medical Institute; Estados UnidosFil: Audetat, Audrey. Howard Hughes Medical Institute; Estados UnidosFil: Pandey, Ahwan. Howard Hughes Medical Institute; Estados UnidosFil: Bonner, Elyzabeth A.. State University Of Colorado Boulder; Estados UnidosFil: Urh, Marjeta. Promega Corporation; Estados UnidosFil: Mendez, Jacqui. Promega Corporation; Estados UnidosFil: Daniels, Danette L.. Promega Corporation; Estados UnidosFil: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Galbraith, Matthew D.. Howard Hughes Medical Institute; Estados UnidosFil: Espinosa, Joaquín M.. Howard Hughes Medical Institute; Estados Unido