Identification of highly selective SIK1/2 inhibitors that modulate innate immune activation and suppress intestinal inflammation.

The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.

Beam Steering 3D Printed Dielectric Lens Antennas for Millimeter-Wave and 5G Applications.

Two types of cost-efficient antennas based on dielectric gradient index dielectric lens have been designed for 5G applications at 28 GHz. The first is a linearly polarized flat lens antenna (LP-FLA) for terrestrial 5G communications. The second is a novel circularly polarized stepped lens antenna (CP-SLA) for 5G satellite services. An efficient design method is presented to optimize and conform the lens topology to the radiation pattern coming from the antenna feeder. The LP-FLA is fed by a traditional linearly polarized pyramidal horn antenna (PHA). The CP-SLA is fed by an open-ended bow-tie waveguide cavity (BCA) antenna. This cavity feeder (BCA), using cross-sections with bow-tie shapes, allows having circular polarization at the desired frequency bandwidth. The two types of presented antennas have been manufactured in order to verify their performance by an easy, low-cost, three-dimensional (3D) printing technique based on stereolithography. The peak realized gain value for the flat (LP-FLA) and stepped (CP-SLA) lens antennas have been increased at 28 GHz to 25.2 and 24.8 dBi, respectively, by disposing the lens structures at the appropriated distance from the feeders. Likewise, using an array of horns (PHA) or open-ended bow-tie waveguide cavity (BCA) antenna feeders, it is possible to obtain a maximum steering angle range of 20° and 35°, for a directivity over 15 dBi and 10 dBi, in the planar and stepped lens antennas, respectively.

Total synthesis and isolation of citrinalin and cyclopiamine congeners.

Many natural products that contain basic nitrogen atoms--for example alkaloids like morphine and quinine-have the potential to treat a broad range of human diseases. However, the presence of a nitrogen atom in a target molecule can complicate its chemical synthesis because of the basicity of nitrogen atoms and their susceptibility to oxidation. Obtaining such compounds by chemical synthesis can be further complicated by the presence of multiple nitrogen atoms, but it can be done by the selective introduction and removal of functional groups that mitigate basicity. Here we use such a strategy to complete the chemical syntheses of citrinalin B and cyclopiamine B. The chemical connections that have been realized as a result of these syntheses, in addition to the isolation of both 17-hydroxycitrinalin B and citrinalin C (which contains a bicyclo[2.2.2]diazaoctane structural unit) through carbon-13 feeding studies, support the existence of a common bicyclo[2.2.2]diazaoctane-containing biogenetic precursor to these compounds, as has been proposed previously.

Function and Structure of MalA/MalA', Iterative Halogenases for Late-Stage C-H Functionalization of Indole Alkaloids.

Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA' catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogues were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA' in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases and provides new insights into a potentially unique reaction mechanism.

A transistor model for the cystic fibrosis transmembrane conductance regulator.

In this paper we present a transistor circuit model for cystic fibrosis transmembrane conductance regulator (CFTR) that seeks to map the functional form of CFTR both in wild type and mutants. The circuit architecture is configured so that the function, and as much as possible the form, faithfully represents what is known about CFTR from cryo-electron microscopy and molecular dynamics. The model is a mixed analog-digital topology with an AND gate receiving the input from two separate ATP-nucleotide-binding domain binding events. The analog portion of the circuit takes the output from the AND gate as its input. The input to the circuit model and its noise characteristics are extracted from single-channel patch-clamp experiments. The chloride current predicted by the model is then compared with single-channel patch-clamp recordings for wild-type CFTR. We also consider the patch-clamp recordings from CFTR with a G551D point mutation, a clinically relevant mutant that is responsive to therapeutic management. Our circuit model approach enables bioengineering approaches to CFTR and allows biophysicists to use efficient circuit simulation tools to analyze its behavior.

GPR15 expressed in T lymphocytes from RA patients is involved in leukocyte chemotaxis to the synovium.

The rheumatoid arthritis (RA) inflammatory process occurs in the joints where immune cells are attracted into the synovium to promote remodeling and tissue damage. GPR15 is a G protein-coupled receptor (GPCR) located on chromosome 3 and has similarity in its sequence with chemokine receptors. Recent evidence indicates that GPR15 may be associated with modulation of the chronic inflammatory response. We evaluated the expression of GPR15 and GPR15L in blood and synovial tissue samples from RA patients, as well as to perform a functional migration assay in response to GPR15L. The expression of GPR15 and c10orf99/gpr15l mRNA was analyzed by RT-qPCR. Samples of synovial fluid and peripheral blood were analyzed for CD45+CD3+CD4+GPR15+ and CD45+CD3+CD8+GPR15+ T cell frequency comparing RA patients versus control subjects by flow cytometry. Migration assays were performed using PBMCs isolated from these individuals in response to the synthetic GPR15 ligand. Statistical analysis included Kruskal-Wallis test, T-test, or Mann-Whitney U test, according to data distribution. A higher expression in the mRNA for GPR15 was identified in early RA subjects. The frequencies of CD4+/CD8+ GPR15+ T lymphocytes are higher in RA patients comparing with healthy subjects. Also, the frequency CD4+/CD8+ GPR15+ T lymphocytes are higher in synovial fluid of established RA patients comparing with OA patients. GPR15 and GPR15L are present in the synovial tissue of RA patients and GPR15L promotes migration of PBMCs from RA patients and healthy subjects. Our results suggest that GPR15/GPR15L have a pathogenic role in RA and their antagonizing could be a therapeutic approach in RA.

Novel Reagent Space: Identifying Unorderable but Readily Synthesizable Building Blocks.

Drug discovery building blocks available commercially or within an internal inventory cover a diverse range of chemical space and yet describe only a tiny fraction of all chemically feasible reagents. Vendors will eagerly provide tools to search the former; there is no straightforward method of mining the latter. We describe a procedure and use case in assembling chemical structures not available for purchase but that could likely be synthesized in one robust chemical transformation starting from readily available building blocks. Accessing this vast virtual chemical space dramatically increases our curated collection of reagents available for medicinal chemistry exploration and novel hit generation, almost tripling the number of those with 10 or fewer atoms.

Waveguide Manufacturing Technologies for Next-Generation Millimeter-Wave Antennas.

Some recent waveguide-based antennas are presented in this paper, designed for the next generation of communication systems operating at the millimeter-wave band. The presented prototypes have been conceived to be manufactured using different state-of-the-art techniques, involving subtractive and additive approaches. All the designs have used the latest developments in the field of manufacturing to guarantee the required accuracy for operation at millimeter-wave frequencies, where tolerances are extremely tight. Different designs will be presented, including a monopulse antenna combining a comparator network, a mode converter, and a spline profile horn; a tunable phase shifter that is integrated into an array to implement reconfigurability of the main lobe direction; and a conformal array antenna. These prototypes were manufactured by diverse approaches taking into account the waveguide configuration, combining parts with high-precision milling, electrical discharge machining, direct metal laser sintering, or stereolithography with spray metallization, showing very competitive performances at the millimeter-wave band till 40 GHz.

Crystal symmetry determination in electron diffraction using machine learning.

Electron backscatter diffraction (EBSD) is one of the primary tools for crystal structure determination. However, this method requires human input to select potential phases for Hough-based or dictionary pattern matching and is not well suited for phase identification. Automated phase identification is the first step in making EBSD into a high-throughput technique. We used a machine learning-based approach and developed a general methodology for rapid and autonomous identification of the crystal symmetry from EBSD patterns. We evaluated our algorithm with diffraction patterns from materials outside the training set. The neural network assigned importance to the same symmetry features that a crystallographer would use for structure identification.

A unified strategy to reverse-prenylated indole alkaloids: total syntheses of preparaherquamide, premalbrancheamide, and (+)-VM-55599.

A full account of our studies toward reverse-prenylated indole alkaloids that contain a bicyclo[2.2.2]core is described. A divergent route is reported which has resulted in the synthesis of preparaherquamide, (+)-VM-55599, and premalbrancheamide. An intramolecular Dieckmann cyclization between an enolate and isocyanate was used to forge the bicyclo[2.2.2]diazaoctane core that is characteristic of these molecules. The pentacyclic indole scaffold was constructed through a one-pot Hofmann rearrangement followed by Fischer indole synthesis. The utilization of our previously reported indole peripheral functionalization strategy also led to natural products including malbrancheamides B, C, stephacidin A, notoamides F, I and R, aspergamide B, and waikialoid A. Ultimately, the divergent route that we devised provided access to a wide range of prenylated indole alkaloids that are differently substituted on the cyclic amine core.

Bioinspired chemical synthesis of monomeric and dimeric stephacidin A congeners.

Stephacidin A and its congeners are a collection of secondary metabolites that possess intriguing structural motifs. They stem from unusual biosynthetic sequences that lead to the incorporation of a prenyl or reverse-prenyl group into a bicyclo[2.2.2]diazaoctane framework, a chromene unit or the vestige thereof. To complement biosynthetic studies, which normally play a significant role in unveiling the biosynthetic pathways of natural products, here we demonstrate that chemical synthesis can provide important insights into biosynthesis. We identify a short total synthesis of congeners in the reverse-prenylated indole alkaloid family related to stephacidin A by taking advantage of a direct indole C6 halogenation of the related ketopremalbrancheamide. This novel strategic approach has now made possible the syntheses of several natural products, including malbrancheamides B and C, notoamides F, I and R, aspergamide B, and waikialoid A, which is a heterodimer of avrainvillamide and aspergamide B. Our approach to the preparation of these prenylated and reverse-prenylated indole alkaloids is bioinspired, and may also inform the as-yet undetermined biosynthesis of several congeners.

Unified Approach to Prenylated Indole Alkaloids: Total Syntheses of (-)-17-Hydroxy-Citrinalin B, (+)-Stephacidin A, and (+)-Notoamide I.

A unified strategy for the synthesis of congeners of the prenylated indole alkaloids is presented. This strategy has yielded the first synthesis of the natural product (-)-17-hydroxy-citrinalin B as well as syntheses of (+)-stephacidin A and (+)-notoamide I. An enolate addition to an in situ generated isocyanate was utilized in forging a key bicyclo[2.2.2]diazaoctane moiety, and in this way connected the two structural classes of the prenylated indole alkaloids through synthesis.