Douglas R. Davies

Specific Btk inhibition suppresses B cell– and myeloid cell–mediated arthritis

Brutons tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

Crystal Structure of Interleukin-6 in Complex with a Modified Nucleic Acid Ligand

Background: Traditional aptamers favor polar interactions with protein binding partners. Results: The IL-6·SOMAmer structure reveals an interface rich in hydrophobic interactions that overlap the binding sites of IL-6 receptors. Conclusion: Hydrophobic modifications on DNA scaffolds generate diverse and novel structural motifs. Significance: Synthetic SOMAmers are potent, specific, and chemically versatile ligands with distinct binding properties compared with conventional aptamers. IL-6 is a secreted cytokine that functions through binding two cell surface receptors, IL-6Rα and gp130. Because of its involvement in the progression of several chronic inflammatory diseases, IL-6 is a target of pharmacologic interest. We have recently identified a novel class of ligands called SOMAmers (S low Off-rate Modified Aptamers) that bind IL-6 and inhibit its biologic activity. SOMAmers exploit the chemical diversity of protein-like side chains assembled on flexible nucleic acid scaffolds, resulting in an expanded repertoire of intra- and intermolecular interactions not achievable with conventional aptamers. Here, we report the co-crystal structure of a high affinity SOMAmer (Kd = 0.20 nm) modified at the 5-position of deoxyuridine in a complex with IL-6. The SOMAmer, comprised of a G-quartet domain and a stem-loop domain, engages IL-6 in a clamp-like manner over an extended surface exhibiting close shape complementarity with the protein. The interface is characterized by substantial hydrophobic interactions overlapping the binding surfaces of the IL-6Rα and gp130 receptors. The G-quartet domain retains considerable binding activity as a disconnected autonomous fragment (Kd = 270 nm). A single substitution from our diversely modified nucleotide library leads to a 37-fold enhancement in binding affinity of the G-quartet fragment (Kd = 7.4 nm). The ability to probe ligand surfaces in this manner is a powerful tool in the development of new therapeutic reagents with improved pharmacologic properties. The SOMAmer·IL-6 structure also expands our understanding of the diverse structural motifs achievable with modified nucleic acid libraries and elucidates the nature with which these unique ligands interact with their protein targets.

Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

Identifying Regulators for EAG1 Channels with a Novel Electrophysiology and Tryptophan Fluorescence Based Screen

Background Ether-à-go-go (EAG) channels are expressed throughout the central nervous system and are also crucial regulators of cell cycle and tumor progression. The large intracellular amino- and carboxy- terminal domains of EAG1 each share similarity with known ligand binding motifs in other proteins, yet EAG1 channels have no known regulatory ligands. Methodology/Principal Findings Here we screened a library of small biologically relevant molecules against EAG1 channels with a novel two-pronged screen to identify channel regulators. In one arm of the screen we used electrophysiology to assess the functional effects of the library compounds on full-length EAG1 channels. In an orthogonal arm, we used tryptophan fluorescence to screen for binding of the library compounds to the isolated C-terminal region. Conclusions/Significance Several compounds from the flavonoid, indole and benzofuran chemical families emerged as binding partners and/or regulators of EAG1 channels. The two-prong screen can aid ligand and drug discovery for ligand-binding domains of other ion channels.

Predicting the success of fragment screening by X-ray crystallography.

Fragment screening using X-ray crystallography is a method that can provide direct three-dimensional readouts of the structures of protein-small molecule complexes for lead development and fragment-based drug discovery. With current technology, an amenable crystal form can be screened crystallographically against a library of 1000-2000 fragments in 1-2 weeks. We have performed over a dozen crystallographic screening campaigns using our own compound collection called Fragments of Life™ (FOL). While the majority of our fragment screening campaigns have generated multiple hits, some unexpectedly turned out to be nonproductive, either yielding no bound ligands, or only those thought to be inadequate for lead development. In this chapter, we have attempted to identify one or more parameters which could be used to predict whether a crystallized protein target would be a good candidate for fragment hit discovery. Here, we describe the parameters of crystals from 18 fragment screening campaigns, including six unsuccessful targets. From this analysis, we have concluded that there are no parameters that are absolutely predictive of fragment screening success. However, we do describe a parameter we have termed pocket factor which provides a statistically significant variance between nonproductive targets and productive targets shown to bind fragments. The pocket factor is calculated using a novel method of consensus scoring from three distinct pocket-finding algorithms, and the results may be used to prioritize targets for fragment screening campaigns based on an initial crystal structure.

An ensemble of structures of Burkholderia pseudomallei 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase.

An ensemble of crystal structures are reported for 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase from B. pseudomallei. The structures include two vanadate complexes, revealing the structure of a close analogue of the transition state for phosphate transfer.

Fragment screening of infectious disease targets in a structural genomics environment

Structural genomics efforts have traditionally focused on generating single protein structures of unique and diverse targets. However, a lone structure for a given target is often insufficient to firmly assign function or to drive drug discovery. As part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we seek to expand the focus of structural genomics by elucidating ensembles of structures that examine small molecule-protein interactions for selected infectious disease targets. In this chapter, we discuss two applications for small molecule libraries in structural genomics: unbiased fragment screening, to provide inspiration for lead development, and targeted, knowledge-based screening, to confirm or correct the functional annotation of a given gene product. This shift in emphasis results in a structural genomics effort that is more engaged with the infectious disease research community, and one that produces structures of greater utility to researchers interested in both protein function and inhibitor development. We also describe specific methods for conducting high-throughput fragment screening in a structural genomics context by X-ray crystallography.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

The first crystal structure is reported of a glutaryl-CoA dehydrogenase in the apo state without flavin adenine dinucleotide cofactor bound. Additional structures with small molecules complexed in the catalytic active site were obtained by fragment-based screening.

Iron superoxide dismutases in eukaryotic pathogens: new insights from Apicomplexa and Trypanosoma structures.

Prior studies have highlighted the potential of superoxide dismutases as drug targets in eukaryotic pathogens. This report presents the structures of three iron-dependent superoxide dismutases (FeSODs) from Trypanosoma cruzi, Leishmania major and Babesia bovis. Comparison with existing structures from Plasmodium and other trypanosome isoforms shows a very conserved overall fold with subtle differences. In particular, structural data suggest that B. bovis FeSOD may display similar resistance to peroxynitrite-mediated inactivation via an intramolecular electron-transfer pathway as previously described in T. cruzi FeSOD isoform B, thus providing valuable information for structure-based drug design. Furthermore, lysine-acetylation results in T. cruzi indicate that acetylation occurs at a position close to that responsible for the regulation of acetylation-mediated activity in the human enzyme.