Artificial neural networks enable genome-scale simulations of intracellular signaling

Mammalian cells adapt their functional state in response to external signals in form of ligands that bind receptors on the cell-surface. Mechanistically, this involves signal-processing through a complex network of molecular interactions that govern transcription factor activity patterns. Computer simulations of the information flow through this network could help predict cellular responses in health and disease. Here we develop a recurrent neural network framework constrained by prior knowledge of the signaling network with ligand-concentrations as input and transcription factor-activity as output. Applied to synthetic data, it predicts unseen test-data (Pearson correlation r = 0.98) and the effects of gene knockouts (r = 0.8). We stimulate macrophages with 59 different ligands, with and without the addition of lipopolysaccharide, and collect transcriptomics data. The framework predicts this data under cross-validation (r = 0.8) and knockout simulations suggest a role for RIPK1 in modulating the lipopolysaccharide response. This work demonstrates the feasibility of genome-scale simulations of intracellular signaling. Many diseases are caused by disruptions to the network of biochemical reactions that allow cells to respond to external signals. Here Nilsson et al develop a method to simulate cellular signaling using artificial neural networks to predict cellular responses and activities of signaling molecules

Nuclear magnetic resonance spectroscopy for structural characterization of bioactive compounds

The structural assignment of a new natural product molecule is not only to establish the 3D structure of a compound, but potentially to provide the basis for research in a multitude of disciplines, ultimately generating new therapeutic agents and/or new understanding of disease biology. The development of modern spectroscopic techniques has transformed the structure assignment process, which previously was essentially based on chemical degradation or derivatization followed by partial or total synthesis. Notably, it was only in the specialization era of the spectroscopic structural assignment of natural products that the field of marine natural products chemistry took shape. Today the processes of marine and terrestrial natural product isolation and structural determination are frequently streamlined and expeditious due to the spectacular advances in chromatographic and spectroscopic technologies as well as chemical synthesis. The NMR spectroscopy is a powerful tool in structure elucidation because the properties it displays can be related to the molecular structure. The chemical environment of a particular nucleus is associated with the chemical shift (d, ppm), and the area of a resonance, usually presented as its relative integral, is related to the number of nuclei giving rise to the NMR signal. The interactions between individual nuclei, mediated by electrons in a chemical bond, determine the coupling constant (J, Hz). In this chapter we will present the techniques commonly used, basic concepts, and how they are useful for chemists in the structural elucidation of mainly bioactive marine natural products. Its complex planar structure is determined by 1H and 13C NMR analysis strongly supported by other 1D (DEPT) and 2D (COSY, TOCSY, HSQC/HMQC, HMBC) NMR techniques. The stereochemistry is generally based on NOE experiments (NOE difference, NOESY, and ROESY), 1H–1H and 1H–13C coupling constants, chiral derivatizing agents, and also in empirical procedures comparing the chemical shifts of unknown vicinal and proximal centers with libraries of configurationally known stereomodels. However, the most reliable option to assign all the 3D structure of a marine natural product still is their total synthesis. The use of NMR hyphenated with other chromatographic and spectroscopic techniques and microcoil probes and narrow diameter tube probes for the structural elucidation of bioactive marine natural products, mainly associated with the quantitative NMR determinations, will be also briefly described. The chapter will finish with a description of the structural characterization of several types of marine natural products using all the referred NMR techniques followed by a small reference to the misassignments that still are very common

Bio-orthogonal Fluorescent Labelling of Biopolymers through Inverse-Electron-Demand Diels–Alder Reactions

Bio-ort hogona llabellin gschemes based on inverse-elec tron- deman dDiels–Ald er (IEDDA) cycloa ddition have attracted much attention in chem ical biology recently .The appeal ing features of this reactio n, such as the fast reactio nkinetics, fully bio-ort hogonal nature and high selectiv ity,have helped chem i- cal biologists gain deeper understandi ng of biochemic al pro- cesses at the molecular level.Listing the compo nents and dis- cussing the possib ilities andlimitations of thesereagent s, we provid earecent snapshot of the field of IEDDA -based biomo- lecular manipulatio nwith special focus on fluores cent modula- tion approaches throug hthe use of bio-orthogon alized build- ing blocks. At the end, we discuss challenges that need to be addres sed for further develop ments in order to overcome recent limita tions and to enabl eresearchers to answer biomo - lecular quest ions in more detail

Tetrazine-mediated bioorthogonal prodrug–prodrug activation

The selective and biocompatible activation of prodrugs within complex biological systems remains a key challenge in medical chemistry and chemical biology. Herein we report, for the first time, a dual prodrug activation strategy that fully satisfies the principle of bioorthogonality by the symbiotic formation of two active drugs. This dual and traceless prodrug activation strategy takes advantage of the INVDA chemistry of tetrazines (here a prodrug), generating a pyridazine-based miR21 inhibitor and the anti-cancer drug camptothecin and offers a new concept in prodrug activation.ISSN:2041-6520ISSN:2041-653

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C2-BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).</p

Function oriented synthesis of bioactive marine natural products and their pharmacophore analogues

Natural products play a central role in drug discovery. The Andersen lab focuses its efforts on the isolation and structure elucidation of compounds from the marine environment. Many of these compounds possess biological activity, and often their total synthesis is undertaken, to provide structure-activity relationship (SAR) studies for new pharmacophores, and to provide material to probe in vivo biological effects. Several projects probing the biological activities of natural products and their analogues by synthesis were completed. It has been proposed that small molecule activators of SHIP1 may be used as a novel therapy for hematopoietic malignancies as well as inflammatory disorders. Activation of the SHIP1 enzymatic pathway may also provide an alternative to PI3K inhibition. An SAR study based on the SHIP1 activating marine natural product pelorol was completed. The goal of the SAR study was to construct water-soluble analogues for the purpose of enhancing drug-like properties. The study yielded analogues that were active in vitro and in vivo. Small molecule antagonists of the N-terminal domain (NTD) of the androgen receptor (AR) are an appealing avenue of exploration for treating CRPC, an advanced form of prostate cancer resistant to current therapies. The marine natural products niphatenone A and B represent a novel NTD-AR antagonist pharmacophore. Their total syntheses were completed to aid in structure determination and provide additional material for biological testing. Furthermore, a click chemistry probe was constructed and it was shown that the natural product covalently binds to the NTD of the AR. Small molecule AR antagonists are currently used as a therapeutic treatment for prostate cancer. Studies towards the total synthesis of a terpene marine natural product discovered to be an AR antagonist are described. The biological role of cathepsin K in bone resorption has led to the development of inhibitors of cathepsin K as potential therapeutics to combat osteoporosis. Lichostatinal, a novel peptide-aldehyde natural product isolated from cultures of a terrestrial actinomycete was found to be a potent inhibitor of cathepsin K. Synthetic efforts towards lichostatinal, in order to verify its structure and to provide additional material for biological testing is described.Science, Faculty ofChemistry, Department ofGraduat