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Hydrogen isotope fractionation between source water and lipids is highly variable among different taxonomic groups, and also among different compound classes within individual organisms. This variability results in lipid δ2H values that often span as much as two orders of magnitude more variability than that of environmental waters within typical ecosystems, indicating that lipid δ2H values may provide valuable biochemical and ecological information. These applications of lipid δ2H values remain underexplored.Recent results from algal batch cultures indicate that hydrogen isotope fractionation by cyanobacteria differs significantly compared to eukaryotic algae. In particular, in single species cultures with constant water δ2H values, cyanobacteria tend to produce fatty acids that are slightly 2H-enriched compared to those from most eukaryotic algae, while phytol from cyanobacteria is very 2H-depleted compared to phytol from eukaryotes. This results in larger offsets between the δ2H values of phytol and fatty acids for cyanobacteria than those observed in eukaryotic algae. In order to determine if δ2H offsets between fatty acids and phytol change in freshwater lakes with variable abundance of cyanobacteria, we collected algal biomass from two depths in the water column of Rotsee, a small lake in central Switzerland, every second week from January 2019 to February 2020. During this time the percentage of algal biovolume from cyanobacteria ranged from 0 to 82 %, with two distinct cyanobacterial blooms occurring in July and October.Water isotopes in the lake were relatively stable throughout the year, with water δ2H values varying by < 10 ‰. Lipid δ2H values, on the other hand, displayed extreme variability throughout the year. Palmitic acid (C 16:0) δ2H values varied by nearly 100 ‰ (–282 to –192 ‰), while those of phytol varied by more than 200 ‰ (–417 to –168 ‰). Consistent with expectations based on the results of cultures of single algal species, cyanobacterial blooms were characterized by larger offsets between the δ2H values of palmitic acid and phytol, and these offsets were positively correlated with the percentage of total algal biovolume attributable to cyanobacteria (R2 = 0.29; p < 0.01). These results suggest that hydrogen isotope offsets between palmitic acid and phytol in sediments have the potential to be developed as proxies for past cyanobacterial blooms, and demonstrate that hydrogen isotopes of lipids in the geologic record that are produced by many different types of aquatic organisms are more likely to be driven by ecological changes rather than changes in water isotopes.

Currently, Holocene paleoclimate research shows discrepancies in the timing and extent of the so-called Holocene “climate optimum” (1). To better understand this phenomenon in the alpine region, we examine the mean annual air temperature (MAT) record based on the distribution of Glycerol Dialkyl Glycerol Tetraethers (GDGTs) in a 14-m long sediment core from Lake Rot, Switzerland. This small eutrophic monomictic lake is characterized by a seasonally anoxic hypolimnion. An age model based on 20 calibrated 14C dates shows that the top 10 m of sediments reflect the early, middle, and late Holocene (10 cal. ka BP to recent). To constrain environmental changes, we also look at total organic carbon (TOC), total inorganic carbon (TIC), total nitrogen (TN), and bulk organic matter δ13C and δ15N (n = 300). These indices give insight into the sources of organic matter in Lake Rot sediments. A stable and dominantly in-situ produced lacustrine source of organic matter is indicated by the range in C/N values (4-17) and d15N values (-0.37-5.84). Increasing TOC and δ13C values during the early Holocene (around 10 cal. ka BP), likely reflect elevated primary production in the lake during postglacial climate warming. Subsequently, high TIC values indicate a period with high calcite precipitation (10-8 cal. ka BP). Between 8-1.5 cal. ka BP, high TOC and very low TIC values indicate a dramatic change in the system, reflecting a higher production and/or conservation of organic matter. After this period, TOC decreases, showing a last increase in the top 50cm of the core, presenting signs of eutrophication. Lake Rot thus has experienced large changes in the last 10ka.From a subset of 63 samples, GDGTs are analysed to reconstruct MAT using the methylation index of brGDGTs (MBT’5ME). Using a lake calibration (2), reconstructed average MAT is 8.4℃ (RMSE = 2.1℃). The absence of large temperature changes during the Holocene highlights that the MBT’5ME-based reconstructed temperatures are not influenced by the large changes in water chemistry recorded by the bulk TOC and TIC values. Instead, the temperature reconstruction reflects stable Holocene temperature ranges, presenting no expressed “climate optimum” in this core. The most recent reconstructed temperature of 9.7℃ resembles actual measured MAT (10.7℃).Based on our results, the isoprenoid GDGT TEX86 is not applicable for the reconstruction of temperature in Lake Rot. This matches a recent study of perialpine lakes where the successful application of TEX86 was suggested to be limited to deep lakes (>100 m) (3). In addition, we will discuss whether production of in-situ brGDGTs in the water column and seasonality influence the sediment temperature record, as proposed by the authors and other studies (2,4). 1: Herzschuh et al., (2022). EGUsphere, 1-23.2: Russell et al., (2018). Organic Geochemistry, 117, 56-69.3: Damsté et al., (2022). Quaternary Science Reviews, 277, 107352.4: Loomis et al., (2014). Geochimica et Cosmochimica Acta, 144, 173-187.

Agriculture relies heavily on the use of plastic mulch films, which increase crop yields and lower water demands. Research has shown that agricultural mulch film may be transported to aquatic environments. We tested the mineralization of soil-biodegradable agricultural mulch films in freshwater lake sediments. Two types of commercial soil-biodegradable mulch films were incubated within lake sediment cores, and the production of CO2 and CH4 was followed over time. After the 40-week incubation period, films were visually intact and showed no signs of mineralization. Gas analyses showed no additional production of either CO2 or CH4 in the degradable film incubations, compared to control or PE plastic incubations. We conclude that the tested soil-biodegradable mulch films have a low biodegradability in lake sediments, likely reflecting that the lake sediment lacks active microbial degraders. Our results highlight the importance of preventing transport of soil-biodegradable mulch films from agricultural soils to surrounding aquatic environments.

Addressing a quest by Gupta et al. (in: Proceedings of the 41st international colloquium on automata, languages, and programming (ICALP 2014), vol 8572 of LNCS. Springer, pp 563–575, 2014), we provide a first, comprehensive study of finding a short s–t path in the multistage graph model, referred to as the Multistages–tPath problem. Herein, given a sequence of graphs over the same vertex set but changing edge sets, the task is to find short s–t paths in each graph (“snapshot”) such that in the found path sequence the consecutive s–t paths are “similar”. We measure similarity by the size of the symmetric difference of either the vertex set (vertex-similarity) or the edge set (edge-similarity) of any two consecutive paths. We prove that these two variants of Multistages–tPath are already $${\text {NP}}$$ NP -hard for an input sequence of only two snapshots and maximum vertex degree four. Motivated by this fact and natural applications of this scenario e.g. in traffic route planning, we perform a parameterized complexity analysis. Among other results, for both variants, vertex- and edge-similarity, we prove parameterized hardness ($${\text {W[1]}}$$ W[1] -hardness) regarding the parameter path length (solution size). As a further conceptual investigation, we then modify the multistage model by asking for dissimilar consecutive paths. As one of the main technical results (employing so-called representative sets known from non-temporal settings), we prove that dissimilarity allows for fixed-parameter tractability for the parameter solution size, contrasting with our W[1]-hardness proof of the corresponding similarity case. We also provide partially positive results concerning efficient and effective data reduction (kernelization).

Carbohydrates are a ubiquitous constituent of living organisms and an important contributor to global sedimentary carbon pools. Yet, the factors that control the pool size and degradation of sedimentary carbohydrates are not well understood. Here, we investigated carbohydrate cycling over a complete Holocene sedimentary succession in high-altitude, meromictic Lake Cadagno (Switzerland). This succession includes sedimentary records of a stepwise evolving lake redox history from oxic, hypoxic to euxinic anoxic, as well as intercalated layers of lacustrine and event deposits. Event deposits differ from lacustrine layers in organic carbon content, stable isotope signatures, carbohydrate contents, and carbohydrate macromolecular compositions (pyrolysis gas/mass spectrometry) indicating significant terrestrial inputs. However, past changes in redox conditions, implied by redox-sensitive elements (e.g. Mn, Fe, Mo, and S), are not reflected in carbon isotope and carbohydrate data. Carbohydrate contributions are stable, or even increase, with sediment age and show compound-specific variations. Certain carbohydrate pyrolysis products of likely aquatic origin, such as levoglucosan, increase in percentage with sediment age, whereas others, such as furaldehyde, decrease with age. On the other hand, pyrolysis products that are of likely terrestrial origin, such as 3-acetamido-methylfuran, decrease strongly with age. In contrast to carbohydrate macromolecules, gas chromatography with flame-ionization detection analyses showed no clear changes in total hydrolyzed carbohydrate monomer compositions in relation to sediment age or between aquatic- and terrestrial-dominated layers. Our results indicate that carbohydrate degradation in sediments is mainly controlled at the macromolecular level. Notably, our findings suggest that carbohydrates can be well-preserved over the entire Holocene in lake sediments and that aquatic carbohydrates are in some cases selectively preserved over their terrestrial counterparts in these sediments.

Past temperature records are key tools for inferring climate dynamics and provide empirical data for testing climate models to improve our mechanistic understanding of natural climate variability. Unfortunately, very few quantitative records of pre-historic continental temperatures exist in Europe. Moreover, existing paleothermometers mainly provide mean annual or warm season temperatures, limiting our understanding of climate variability during the transitional seasons and winter. Alkenones are temperature-sensitive lipids produced by Isochrysidales algae, which have been used for decades to reconstruct quantitative changes in sea-surface temperatures. In lakes, they are not ubiquitous, but they have been increasingly reported in both saline and freshwater lakes worldwide, suggesting that there is great potential for alkenone-based paleotemperature reconstructions in lacustrine settings. Lacustrine alkenones have already been successfuly used to reconstruct paleotemperatures in high-latitude lakes. Depending on the timing of ice-out, they record winter/spring or summer temperatures. In our study, we found that a significant number of Swiss lakes contain lacustrine alkenones. Other studies in mid-latitude European lakes suggest that the peak of alkenone production occurs in spring. The monitoring of Lake St Moritz, an alpine lake in the South East of Switzerland, will allow determining the seasonality of alkenone production in mid-latitute high altitude lakes. Combining genetic analyses and the monitoring of physico-chemical parameters will provide more information about the ecology of the alkenone producers. Our first results suggest that we will be able to improve the understanding of alkenone production in freshwater lakes and to develop the first spring lake temperature reconstruction in Switzerland that extends beyond existing historical records.

Following the increasing interest and adoption of FaaS systems, benchmarking frameworks for determining non-functional properties have also emerged. While existing (microbenchmark) frameworks only evaluate single aspects of FaaS platforms, a more holistic, application-driven approach is still missing. In this paper, we design and present BeFaaS, an extensible application-centric benchmarking framework for FaaS environments that focuses on the evaluation of FaaS platforms through realistic and typical examples of FaaS applications. BeFaaS includes a built-in e-commerce benchmark, is extensible for new workload profiles and new platforms, supports federated benchmark runs in which the benchmark application is distributed over multiple providers, and supports a fine-grained result analysis. Our evaluation compares three major FaaS providers in single cloud provider setups and shows that BeFaaS is capable of running each benchmark automatically with minimal configuration effort and providing detailed insights for each interaction., Accepted for publication in Proc. of IEEE International Conference on Cloud Engineering 2021 (IC2E'21)

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.

We generalize the notion of Lagrangian subspaces to self-orthogonal subspaces with respect to a (skew-) symmetric form, thus characterizing (skew-)self-adjoint and unitary operators by means of self-orthogonal subspaces. By orthogonality preserving mappings, these characterizations can be transferred to abstract boundary value spaces of (skew-)symmetric operators. Introducing the notion of boundary systems we then present a unified treatment of different versions of boundary triples and related concepts treated in the literature. The application of the abstract results yields a description of all (skew-)self-adjoint realizations of Laplace and first derivative operators on graphs.

We consider metric graphs with a uniform lower bound on the edge lengths but no further restrictions. We discuss how to describe every local self-adjoint Laplace operator on such graphs by boundary conditions in the vertices given by projections and self-adjoint operators. We then characterize the lower bounded self-adjoint Laplacians and determine their associated quadratic form in terms of the operator families encoding the boundary conditions.

A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and pharmacodynamic properties and potential toxicological liabilities. Early stage absorption, distribution, metabolism, and excretion assays were employed to ensure the incorporation of druglike properties resulting in the selection of several compounds with good activity in a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory development candidate JNJ-28312141 (23, FMS IC(50) = 0.69 nM, cell assay IC(50) = 2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has entered phase I clinical trials.

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

We construct an expansion in generalized eigenfunctions for Schrodinger operators on metric graphs. We require rather minimal assumptions concerning the graph structure and the boundary conditions at the vertices.

An anti-inflammatory 1,2,4-phenylenetriamine-containing series of FMS inhibitors with a potential to form reactive metabolites was transformed into a series with equivalent potency by incorporation of carbon-based replacement groups. Structure-based modeling provided the framework to efficiently effect this transformation and restore potencies to previous levels. This optimization removed a risk factor for potential idiosyncratic drug reactions.

The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted arylamides. Compound 8 (FMS kinase IC(50)=0.0008 microM) served as a proof-of-concept candidate in a collagen-induced model of arthritis in mice.

A series of 3,4,6-substituted 2-quinolones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). The fully optimized compound, 4-(4-ethyl-phenyl)-3-(2-methyl-3H-imidazol-4-yl)-2-quinolone-6-carbonitrile 21b, has an IC(50) of 2.5 nM in an in vitro assay and 5.0 nM in a bone marrow-derived macrophage cellular assay. Inhibition of FMS signaling in vivo was also demonstrated in a mouse pharmacodynamic model.

An efficient synthesis of compound 1 featuring a novel sequential Friedel–Crafts alkylation strategy to construct the 4-aryl-tetrahydroisoquinoline core structure has been developed. Resolution with (d/l)-di-p-toluoyl-tartaric acid is utilized to provide the enantiomerically pure material. Overall, the route is concise and amenable for large-scale synthesis.

A series of novel 4-aryl-1,2,3,4-tetrahydroisoquinoline-based histamine H(3) ligands that also have serotonin reuptake transporter inhibitor activity is described. The synthesis, in vitro biological data, and select pharmacokinetic data for these novel compounds are discussed.

The 1,4-benzodiazepine-2,5-dione is a suitable template to disrupt the interaction between p53 and Hdm2. The development of an enantioselective synthesis disclosed the stereochemistry of the active enantiomer. An in vitro p53 peptide displacement assay identified active compounds. These activities were confirmed in several cell-based assays including induction of the p53 regulated gene (PIG-3) and caspase activity.

[reaction: see text] A single-step process for the preparation of 2-amino-7-chlorothiazolo[5,4-d]pyrimidines, 2, was achieved by the reaction of the commercially available 4,6-dichloro-5-aminopyrimidine 1 with isothiocyanates. This mild reaction accommodates a variety of functionalized isothiocyanates and proceeds in good to excellent yields. The utility of such intermediates is exemplified by subsequent reaction with alkyl or arylamine nucleophiles to afford novel, differentially functionalized 2,7-diaminothiazolo[5,4-d]pyrimidines, 3.

HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

Background: Arrestins are responsible for the desensitization of many sequence-divergent G protein-coupled receptors. They compete with G proteins for binding to activated phosphorylated receptors, initiate receptor internalization, and activate additional signaling pathways. Results: In order to understand the structural basis for receptor binding and arrestin's function as an adaptor molecule, we determined the X-ray crystal structure of two truncated forms of bovine β-arrestin in its cytosolic inactive state to 1.9 A. Mutational analysis and chimera studies identify the regions in β-arrestin responsible for receptor binding specificity. β-arrestin demonstrates high structural homology with the previously solved visual arrestin. All key structural elements responsible for arrestin's mechanism of activation are conserved. Conclusions: Based on structural analysis and mutagenesis data, we propose a previously unappreciated part in β-arrestin's mode of action by which a cationic amphipathic helix may function as a reversible membrane anchor. This novel activation mechanism would facilitate the formation of a high-affinity complex between β-arrestin and an activated receptor regardless of its specific subtype. Like the interaction between β-arrestin's polar core and the phosphorylated receptor, such a general activation mechanism would contribute to β-arrestin's versatility as a regulator of many receptors.

Arrestins quench the signaling of a wide variety of G protein-coupled receptors by virtue of high-affinity binding to phosphorylated activated receptors. The high selectivity of arrestins for this particular functional form of receptor ensures their timely binding and dissociation. In a continuing effort to elucidate the molecular mechanisms responsible for arrestin's selectivity, we used the visual arrestin model to probe the functions of its N-terminal beta-strand I comprising the highly conserved hydrophobic element Val-Ile-Phe (residues 11-13) and the adjacent positively charged Lys(14) and Lys(15). Charge elimination and reversal in positions 14 and 15 dramatically reduce arrestin binding to phosphorylated light-activated rhodopsin (P-Rh*). The same mutations in the context of various constitutively active arrestin mutants (which bind to P-Rh*, dark phosphorylated rhodopsin (P-Rh), and unphosphorylated light-activated rhodopsin (Rh*)) have minimum impact on P-Rh* and Rh* binding and virtually eliminate P-Rh binding. These results suggest that the two lysines "guide" receptor-attached phosphates toward the phosphorylation-sensitive trigger Arg(175) and participate in phosphate binding in the active state of arrestin. The elimination of the hydrophobic side chains of residues 11-13 (triple mutation V11A, I12A, and F13A) moderately enhances arrestin binding to P-Rh and Rh*. The effects of triple mutation V11A, I12A, and F13A in the context of phosphorylation-independent mutants suggest that residues 11-13 play a dual role. They stabilize arrestin's basal conformation via interaction with hydrophobic elements in arrestin's C-tail and alpha-helix I as well as its active state by interactions with alternative partners. In the context of the recently solved crystal structure of arrestin's basal state, these findings allow us to propose a model of initial phosphate-driven structural rearrangements in arrestin that ultimately result in its transition into the active receptor-binding state.

Visual arrestin is the protein responsible for rapid quenching of G-protein-coupled receptor signaling. Arrestin exists as a latent inhibitor which must be 'activated' upon contact with a phosphorylated receptor. X-ray crystal structures of visual arrestin exhibit a tetrameric arrangement wherein an asymmetric dimer with an extensive interface between conformationally different subunits is related to a second asymmetric dimer by a local two-fold rotation axis. To test the biological relevance of this molecular organization in solution, we carried out a sedimentation equilibrium analysis of arrestin at both crystallographic and physiological protein concentrations. While the tetrameric form can exist at the high concentrations used in crystallography experiments, we find that arrestin participates in a monomer/dimer equilibrium at concentrations more likely to be physiologically relevant. Solution interaction analysis of a proteolytically modified, constitutively active form of arrestin shows diminished dimerization. We propose that self-association of arrestin may provide a mechanism for regulation of arrestin activity by (i) ensuring an adequate supply for rapid quenching of the visual signal and (ii) limiting the availability of active monomeric species, thereby preventing inappropriate signal termination.

Visual arrestin quenches light-induced signaling by binding to light-activated, phosphorylated rhodopsin (P-Rh*). Here we present structure-function data, which in conjunction with the refined crystal structure of arrestin (Hirsch, J. A., Schubert, C., Gurevich, V. V., and Sigler, P. B. (1999)Cell, in press), support a model for the conversion of a basal or “inactive” conformation of free arrestin to one that can bind to and inhibit the light activated receptor. The trigger for this transition is an interaction of the phosphorylated COOH-terminal segment of the receptor with arrestin that disrupts intramolecular interactions, including a hydrogen-bonded network of buried, charged side chains, referred to as the “polar core.” This disruption permits structural adjustments that allow arrestin to bind to the receptor. Our mutational survey identifies residues in arrestin (Arg175, Asp30, Asp296, Asp303, Arg382), which when altered bypass the need for the interaction with the receptor’s phosphopeptide, enabling arrestin to bind to activated, nonphosphorylated rhodopsin (Rh*). These mutational changes disrupt interactions and substructures which the crystallographic model and previous biochemical studies have shown are responsible for maintaining the inactive state. The molecular basis for these disruptions was confirmed by successfully introducing structure-based second site substitutions that restored the critical interactions. The nearly absolute conservation of the mutagenically sensitive residues throughout the arrestin family suggests that this mechanism is likely to be applicable to arrestin-mediated desensitization of most G-protein-coupled receptors.

G protein–coupled signaling is utilized by a wide variety of eukaryotes for communicating information from the extracellular environment. Signal termination is achieved by the action of the arrestins, which bind to activated, phosphorylated G protein–coupled receptors. We describe here crystallographic studies of visual arrestin in its basal conformation. The salient features of the structure are a bipartite molecule with an unusual polar core. This core is stabilized in part by an extended carboxy-terminal tail that locks the molecule into an inactive state. In addition, arrestin is found to be a dimer of two asymmetric molecules, suggesting an intrinsic conformational plasticity. In conjunction with biochemical and mutagenesis data, we propose a molecular mechanism by which arrestin is activated for receptor binding.

The first example of a monocyano-substituted 2-carboxyimidazole is reported. A synthesis of potassium 4-cyano-1-{[2-(trimethylsilyl)ethoxy]methyl} -1 H-imidazole-2-carboxylate (2) is demonstrated where the carboxylate group is introduced via bromine-magnesium exchange on a SEM-protected cyanoimidazole followed by reaction with ethyl cyanoformate. The synthesis includes the equilibration of a regioisomeric mixture of SEM-protected imidazoles to give a single product.

Structure-activity relationship (SAR) studies on a highly potent series of arylamide FMS inhibitors were carried out with the aim of improving FMS kinase selectivity, particularly over KIT. Potent compound 17r (FMS IC50 0.7 nM, FMS cell IC50 6.1 nM) was discovered that had good PK properties and a greater than fivefold improvement in selectivity for FMS over KIT kinase in a cellular assay relative to the previously reported clinical candidate 4. This improved selectivity was manifested in vivo by no observed decrease in circulating reticulocytes, a measure of bone safety, at the highest studied dose. Compound 17r was highly active in a mouse pharmacodynamic model and demonstrated disease-modifying effects in a dose-dependent manner in a strep cell wall-induced arthritis model of rheumatoid arthritis in rats.

A class of potent inhibitors of colony-stimulating factor-1 receptor (CSF-1R or FMS), as exemplified by 8 and 21, was optimized to improve pharmacokinetic and pharmacodynamic properties and potential toxicological liabilities. Early stage absorption, distribution, metabolism, and excretion assays were employed to ensure the incorporation of druglike properties resulting in the selection of several compounds with good activity in a pharmacodynamic screening assay in mice. Further investigation, utilizing the type II collagen-induced arthritis model in mice, culminated in the selection of anti-inflammatory development candidate JNJ-28312141 (23, FMS IC(50) = 0.69 nM, cell assay IC(50) = 2.6 nM). Compound 23 also demonstrated efficacy in rat adjuvant and streptococcal cell wall-induced models of arthritis and has entered phase I clinical trials.

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

During efforts to improve the bioavailability of FMS kinase inhibitors 1 and 2, a series of saturated and aromatic 4-heterocycles of reduced basicity were prepared and evaluated in an attempt to also improve the cardiovascular safety profile over lead arylamide 1, which possessed ion channel activity. The resultant compounds retained excellent potency and exhibited diminished ion channel activity.

A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating rheumatoid arthritis and other chronic inflammatory diseases. Structure-based optimization of the lead amide analogue 10 led to hydroxamate analogue 37, which possessed excellent potency and an improved pharmacokinetic profile. During the chronic phase of streptococcal cell wall-induced arthritis in rats, compound 37 (10, 3, and 1 mg/kg) was highly effective at reversing established joint swelling. In an adjuvant-induced arthritis model in rats, 37 prevented joint swelling partially at 10 mg/kg. In this model, osteoclastogenesis and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation. These data underscore the potential of FMS inhibitors to prevent erosions and reduce symptoms in rheumatoid arthritis.

A series of pyrimidinopyridones has been designed, synthesized and shown to be potent and selective inhibitors of the FMS tyrosine kinase. Introduction of an amide substituent at the 6-position of the pyridone core resulted in a significant potency increase. Compound 24 effectively inhibited in vivo LPS-induced TNF in mice greater than 80%.

A novel series of cholecystokinin-2 receptor (CCK-2R) antagonists has been identified, as exemplified by anthranilic sulfonamide 1 (pK(i)=7.6). Pharmacokinetic and stability studies indicated that this series of compounds suffered from metabolic degradation, and that both the benzothiadiazole and piperidine rings were rapidly oxidized by liver enzymes. A combination of synthesis, computational methods, (1)H NMR conformational studies, and X-ray crystallographic analyses were applied to elucidate key pharmacophore elements, and to discover analogs with improved pharmacokinetic profiles, and high receptor binding affinity and selectivity.

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.

Small molecule antagonists of protein-protein interactions represent a particular challenge for pharmaceutical discovery. One approach to finding molecules that can disrupt these interactions is to seek mimics of common protein structure motifs. We present an analysis of how molecules based on the 1,4-benzodiazepine-2,5-dione scaffold serve to mimic the side-chains presented by the hydrophobic face of two turns of an alpha-helix derived from the tumor suppressor protein p53, and thus antagonize the HDM2-p53 protein-protein binding interaction.

Guided by structure-based drug design, modification of the 1,4-benzodiazepin-2,5-dione lead compound 1 resulted in the discovery of 19, a potent and orally bioavailable antagonist of the HDM2-p53 protein-protein interaction (FP IC50 = 0.7 microM, F approximately 100%).

MAPK-activated protein kinase-2 (MAPKAPK2) regulates the synthesis of tumor necrosis factor and other cytokines and is a potential drug target for inflammatory diseases. Five protein constructs were produced in 4–10 mg quantities per liter of culture media using baculovirus-infected insect cells and characterized for kinase activity, thermal stability, and ligand-binding affinity. Compared to construct 1–370, removal of the C-terminal autoinhibitory peptide in 1–338 resulted in a destabilized but partially active nonphosphorylated enzyme; phosphorylation of 1–338 by p38α further increased activity 12-fold. A putative constitutively active mutant, 1–370/T222E/T334E, was 6.3-fold less active than phosphorylated 1–370. ThermoFluor, an equilibrium ligand-binding assay, was used to measure nucleotide analogue affinity for various constructs. Binding of phosphorylated nucleotides was Mg 2+ -dependent. Residues 1–40 were required for high-affinity binding of ADP, ATPγS, staurosporine, and K252a. A mutation M138A rendered 1–370 susceptible to p38-inhibitors SB-203580 and SB-202190 with IC50 values of 17.4 and 14.1 μM, respectively. Taken together, these studies provide information on the mechanism of ligand-binding to MAPKAPK2 that can be used in the search for selective small-molecule inhibitors.

Crystallographic analysis of ligands bound to HDM2 suggested that 7-substituted 1,4-diazepine-2,5-diones could mimic the alpha-helix of p53 peptide and may represent a promising scaffold to develop HDM2-p53 antagonists. To verify this hypothesis, we synthesized and biologically evaluated 5-[(3S)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-7-phenyl-1,4-diazepin-1-yl]valeric acid (10) and 5-[(3S)-7-(2-bromophenyl)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-1,4-diazepin-1-yl]valeric acid (11). Preliminary in vitro testing shows that 10 and 11 substantially antagonize the binding between HDM2 and p53 with an IC(50) of 13 and 3.6 microM, respectively, validating the modeling predictions. Taken together with the high cell permeability of diazepine 11 determined in CACO-2 cells, these results suggest that 1,4-diazepine-2,5-diones may be useful in the treatment of certain cancers.

Fatty Acid Synthase (FASN) is a multi-domain protein that carries out de novo fatty acid (palmitate) synthesis from acetate and malonate in mammalian cells. FASN is up-regulated in cancer cells, providing fatty acid building blocks for rapid cell growth and cell division. Increased FASN expression is correlated with disease progression and poor prognosis in many cancers including prostate, breast, ovary, colon, and lung. FASN has been demonstrated to play an important role in carcinogenesis by protecting cells from apoptosis. Herein we report a new series of potent, selective and orally bioavailable FASN inhibitors. Recent publications disclose several FASN inhibitor chemotypes that share a common pharmacophore, wherein an aromatic group and an acylated cyclic amine are attached to a central scaffold. We postulated that a spirocyclic imidazolinone core would be an acceptable and drug-like scaffold, inspired by the precedent of irbesartan, an approved antihypertensive drug in which a spirocyclopentyl-imidazolinone core replaces the substituted imidazole ring of losartan, an older approved agent from the same drug class. This hypothesis led to a new spirocyclic imidazolinone based FASN inhibitors. Extensive SAR efforts resulted in FASN inhibitors with potent enzyme and cell activity, selectivity, and oral bioavailability exemplified by JNJ-54302833. JNJ-54302833 is a potent inhibitor of human FASN (IC50 = 28 nM) and also potently inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced medium. This cellular activity can be rescued by addition of palmitate, demonstrating on-target effects. JNJ-54302833 is also potent in many other cells, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposures. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with JNJ-54302833 resulted in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate. This novel series potently inhibits the FASN KR domain (IC50 = 54 nM for JNJ-54302833); specific binding to KR was confirmed by crystal structures.In summary, we have designed and discovered a new series of FASN inhibitors that are potent both in enzyme and in cell proliferation assays, are highly bioavailable, and bind to KR domain. Additionally, palmitate rescue of lipid-reduced cellular activity suggests selectivity and pharmacodynamics studies confirm target engagement. Citation Format: Tianbao Lu, Richard Alexander, Gilles Bignan, James Bischoff, Peter Connolly, Max Cummings, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Bruce Grasberger, Boudewijn Janssens, Donald Ludovici, Lieven Meerpoel, Christophe Meyer, Michael Parker, Danielle Peeters, Carsten Schubert, Karine Smans, Luc Van Nuffel, Peter Vermeulen. Design and synthesis of a series highly potent and bioavailable FASN KR domain inhibitors for cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4747. doi:10.1158/1538-7445.AM2014-4747

Fatty Acid Synthase (FASN) catalyzes the final step in palmitate (PA) synthesis, using acetyl-CoA, malonyl-CoA and NADPH. Most normal tissues express low levels of FASN and rely on uptake of fatty acids (FA) from the diet. It has been proposed that FASN overexpressing tumors including prostate and breast tumors depend on de novo FA synthesis, which is advantageous to tumors by providing lipids for membrane synthesis and increased growth factor receptor expression/signaling in lipid rafts. Overexpression of FASN leads to a higher amount of saturated lipids in membranes which can lead to resistance to cytotoxic chemotherapy. Lastly, NADPH consumption during PA synthesis keeps the redox balance in check. All of the above imply that FASN represents a potential therapeutic target for the treatment of multiple cancer types. At this AACR we report two novel chemical series (posters Connolly et al., Lu et al.). JNJ-53793220 and JNJ-54302833 potently and selectively (< 100nM) inhibit the FASN enzyme and proliferative activity in cells in lipid reduced medium (LRM). Using these, we investigated the underlying hypothesis that tumor cells do not utilize circulating FA and are dependent on de novo synthesis of FA. In a lipid reduced environment many cell lines, particularly of prostate, breast, ovarian or heme origin, proved to be sensitive to JNJ-53793220. However co-administration of PA dose-dependently reversed the anti-proliferative effects. Also androgen driven proliferation of LNCaP cells was potently blocked by JNJ-53793220 (EC50 30 nM), and decreased PSA levels. Both effects were partially rescued by the addition of PA. While the rescue of tumor cells by PA confirmed the on-target activity of the compounds, it also suggested that cancer cells are capable of using external FA. To extend these findings, we screened more than 400 cell lines in lipid containing medium (LCM) for their sensitivity to JNJ-53793220. In LCM sensitivity to FASN inhibition was lower than in LRM conditions. In most, but not all, cases the addition of PA reverted the antiproliferative effects of JNJ-53793220, although target engagement was not reduced in LCM conditions. The EC50 of 14C-acetate incorporation in lipids of ∼30 nM corresponded well with enzymatic and anti-proliferative effects in LRM (27 and 13 nM respectively). Furthermore, growth of pre-established LNCaP xenografts in vivo was not blocked significantly by JNJ-53793220, even though malonyl-CoA levels were increased as expected upon FASN inhibition in the tumor. While circulating lipids in vivo are likely culprits for the lack of efficacy, other factors may play a role as well. In a 3D culture model (poster Vidic et al.) the growth of LNCaP and PC346c spheroids was blocked by JNJ-54302833 (1µM), but growth of PC346c spheroids co-cultured with cancer associated fibroblasts was not inhibited. Taken together our data suggest that the outcome of FASN inhibition is influenced by the tumor environment. Citation Format: Karine A. Smans, Sabine De Breucker, Norbert Esser, Erwin Fraiponts, Ron Gilissen, Ralph Graeser, Boudewijn Janssen, Lieven Meerpoel, Danielle Peeters, Geert Van Hecke, Luc Van Nuffel, Yolanda Chong, Peter Vermeulen, Gilles Bignan, James Bischoff, Peter Connolly, Bruce Grasberger, Tianbao Lu, Donald Ludovici, Carsten Schubert, Michael Parker, Christophe Meyer, Suzana Vidic. Sensitivity of cell lines to Fatty Acid Synthase inhibitors depends on the lipid content in the cellular environment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2014-801